U.S. patent application number 13/764363 was filed with the patent office on 2013-12-26 for protein synthesis modulators.
This patent application is currently assigned to RIB-X PHARMACEUTICALS, INC.. The applicant listed for this patent is Rib-X Pharmaceuticals, Inc.. Invention is credited to Brian T. Wimberly.
Application Number | 20130344562 13/764363 |
Document ID | / |
Family ID | 47632017 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344562 |
Kind Code |
A1 |
Wimberly; Brian T. |
December 26, 2013 |
PROTEIN SYNTHESIS MODULATORS
Abstract
The invention provides a high resolution three-dimensional
structure of cycloheximide, either alone or in association with a
large ribosomal subunit. The invention provides methods for
designing and/or identifying cycloheximide analogs and derivatives
that bind and/or modulate the protein biosynthetic activity of the
ribosome.
Inventors: |
Wimberly; Brian T.;
(Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rib-X Pharmaceuticals, Inc. |
New Haven |
CT |
US |
|
|
Assignee: |
RIB-X PHARMACEUTICALS, INC.
New Haven
CT
|
Family ID: |
47632017 |
Appl. No.: |
13/764363 |
Filed: |
February 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12072980 |
Feb 29, 2008 |
8374794 |
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13764363 |
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10858159 |
Jun 1, 2004 |
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12072980 |
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60475232 |
Jun 2, 2003 |
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Current U.S.
Class: |
435/183 ;
530/300; 530/387.1; 530/395; 536/23.1; 554/1 |
Current CPC
Class: |
G16C 20/50 20190201;
G16B 5/00 20190201; G16B 15/00 20190201; C12P 21/02 20130101 |
Class at
Publication: |
435/183 ; 554/1;
536/23.1; 530/300; 530/395; 530/387.1 |
International
Class: |
G06F 19/12 20060101
G06F019/12 |
Claims
1.-18. (canceled)
19. A method of identifying a candidate molecule, the method
comprising the steps of: (a) providing a molecular model of at
least a portion of a cycloheximide molecule bound to a
cycloheximide binding site of a large subunit of a ribosome; and
(b) using the molecular model to identify a candidate molecule
capable of binding to the cycloheximide binding site; and (c)
producing the candidate molecule identified in step (b).
20. The method of claim 19, wherein the candidate molecule is
capable of interacting stereochemically with the binding site.
21. The method of claim 19, wherein the candidate molecule is
capable of binding specifically to the binding site.
22. The method of claim 19 comprising the additional step of
determining whether the candidate molecule modulates ribosomal
activity.
23. The method of claim 22 comprising the additional step of
modifying the candidate molecule.
24. The method of claim 23 comprising the additional step of
producing the modified candidate molecule.
25. The method of claim 24 comprising the additional step of
determining whether the candidate molecule modulates ribosomal
activity.
26. The method of claim 25 comprising the additional step of
producing the candidate molecule.
27. The method of claim 19, wherein the molecular model is defined
by at least a portion of the atomic co-ordinates recorded on Disk
No. 1 under file name cycloheximide.pdb.
28.-41. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/072,980, filed Feb. 29, 2008 (now U.S. Pat.
No. 8,374,794), which is a continuation of U.S. patent application
Ser. No. 10/858,159, filed Jun. 1, 2004 (now abandoned), and claims
the benefit of the filing date of U.S. provisional patent
application Ser. No. 60/475,232, filed Jun. 2, 2003, the entire
disclosure of each of which is incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
modulators, for example, inhibitors, of ribosome function, and to
methods of making and using such modulators. More particularly, the
invention relates to the antibiotic cycloheximide, the
three-dimensional structure of cycloheximide when associated with a
large subunit of a ribosome, and to methods of making and using
analogs and derivatives of cycloheximide.
BACKGROUND
[0003] The evolution of strains of cells or organisms resistant to
currently effective therapeutic agents is an ongoing medical
problem. For example, the development of cancerous cells resistant
to certain anti-proliferative agents, for example, chemotherapeutic
drugs, has long been recognized as a problem in the oncology field.
Once resistant cells develop, the therapeutic regime must be
modified to introduce other, effective anti-proliferative agents.
Another example of resistance is the development of strains of
microbial, fungal, parasitic and viral pathogens resistant to one
or more anti-infective agents. This problem of resistance to
anti-infective agents is particularly problematic for antibiotic
therapy. Over the past several decades, there has been an increase
in incidence of bacteria that have developed resistance to one or
more antibiotic agents. Because of these resistance problems, there
is a need for new anti-proliferative and anti-infective agents that
are effective against strains of cells or organisms that have
developed resistance to currently available agents.
[0004] In the field of anti-infective agents, a variety of
different agents having antibiotic and other properties have been
developed over the years and approved for use in mammals and other
animals. For example, one such substance is cycloheximide, which is
an antibiotic produced by the streptomycin-producing strains of
Streptomyces griseus. Cycloheximide corresponds to the chemical
formula C.sub.15H.sub.23NO.sub.4 and is also known as
[1S-[1.alpha.(S*),3.alpha.,5.beta.]]-4-[2-(3,5-dimethyl-2-oxocyc-
lohexyl)-2-hydroxyethyl]-2,6-piperidinedione, naramycin A, and
Actidione (See, The Merck Index, 13 Edition, entry 2757, 2001). A
chemical representation for cycloheximide is as shown below.
##STR00001##
[0005] Cycloheximide has long been available for use as an
anti-infective agent active against eukaryotic pathogens, including
fungi. However, cycloheximide is toxic to mammals, and it does not
inhibit bacterial growth. Accordingly, cycloheximide has not been
used to treat bacterial infections, and has only been used to treat
fungal infections on a limited basis. Nevertheless, the structure
of cycloheximide bound to a large ribosomal subunit is useful in
the discovery of new anti-fungal and anti-bacterial agents. For
example, the differences between bacterial, fungal, and human
ribosomes that bind cycloheximide, together with the structure of
cycloheximide bound to a large ribosomal subunit, can guide the
discovery of novel chemical entities useful in treating fungal and
bacterial infections in humans.
[0006] Accordingly, there is still an ongoing need for new analogs
and derivatives of cycloheximide that are effective as
anti-infective, anti-proliferative, or anti-inflammatory
agents.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, upon the
determination of the three-dimensional atomic structure of
cycloheximide in association with the large subunit of a ribosome.
The invention provides methods of using the atomic co-ordinates to
identify cycloheximide analogs and derivatives that selectively
target and/or bind ribosomes, and that preferably act as selective
inhibitors of protein synthesis. The invention also provides a
computer system containing data indicative of atomic co-ordinates
that define at least a portion of the three-dimensional structure
of cycloheximide when disposed within and bound to the large
ribosomal subunit. Each of these aspects of the invention is
discussed in more detail below.
[0008] In one aspect, the invention provides a method of
identifying a molecule that binds to the cycloheximide binding site
of a large ribosomal subunit. The method comprises the steps of:
(a) providing a molecular model, for example, in an electronic
form, of at least a portion of a cycloheximide binding site of a
large subunit of a ribosome; and (b) using the molecular model to
identify a candidate molecule capable of binding to at least a
portion of the cycloheximide binding site of the molecular model.
Optionally, the molecular model in step (a) is created from (i) the
atomic co-ordinates for the Haloarcula marismortui large ribosomal
subunit found on Disk 1 under file name cycloheximide.pdb or under
file name 1JJ2.pdb, or (ii) atomic co-ordinates derived by
molecular modeling from the atomic co-ordinates for the Haloarcula
marismortui large ribosomal subunit found on Disk 1 under file name
cycloheximide.pdb or under file name 1JJ2.pdb.
[0009] The candidate molecules preferably bind specifically to at
least a portion of the cycloheximide binding site of the molecular
model. Furthermore, the candidate molecules stereochemically
interfit and/or have chemical complementarity with the
cycloheximide binding site of the molecular model. The candidate
molecules preferably have a surface a portion of which has a shape
complementary to least a portion of the cycloheximide binding site.
In other words, this complementary relationship is analogous to a
hand having a complementary shape to a glove or a key having a
complementary shape to a lock. In addition to shape, the candidate
molecules have atoms, side chains or groups that are capable of
hydrogen bonding to the atoms, side chains or groups present in the
cycloheximide binding pocket and/or have atoms, side chains or
groups that have a charge opposite in character to the atoms, side
chains, or groups present in the cycloheximide binding pocket.
[0010] The method optionally includes one or more additional steps
of: producing the candidate molecule identified; determining
whether the candidate molecule, when produced, modulates (for
example, induces or reduces) ribosomal activity; identifying a
modified candidate molecule (i.e., a molecule that differs by at
least one atom from the candidate molecule or is an isomer of the
candidate molecule); producing the modified candidate molecule;
determining whether the modified candidate molecule, when produced,
modulates ribosomal activity; and producing the modified candidate
molecule in commercially reasonable amounts for use either alone or
in combination with a pharmaceutically acceptable carrier or
excipient. The candidate molecule and/or the modified candidate
molecule preferably is an antibiotic. Furthermore, the candidate
molecule and/or the modified candidate molecule is capable of
binding to at least a portion of the E-site of the large ribosomal
subunit.
[0011] In another aspect, the invention provides a method of
identifying a candidate molecule. The method comprises the steps
of: (a) providing a molecular model of at least a portion of a
cycloheximide molecule bound to a cycloheximide binding site of a
large subunit of a ribosome; and (b) using the molecular model to
identify a candidate molecule capable of binding to the
cycloheximide binding site. The molecular model is created from at
least a portion of the atomic co-ordinates recorded on Disk No. 1
under file name cycloheximide.pdb or from atomic co-ordinates
derived by molecular modeling, for example, homology modeling
and/or molecular replacement, from the atomic co-ordinates recorded
on Disk No. 1 under file name cycloheximide.pdb.
[0012] The candidate molecules preferably bind specifically to at
least a portion of the cycloheximide binding site of the molecular
model. Furthermore, the candidate molecules stereochemically
interfit and/or have chemical complementarity with the
cycloheximide binding site of the molecular model.
[0013] The method optionally includes one or more additional steps
of producing the identified candidate molecule; determining whether
the candidate molecule, when produced, modulates (for example,
induces or reduces) ribosomal activity; identifying a modified
candidate molecule (i.e., a molecule that differs by at least one
atom from the candidate molecule or that is an isomer of the
candidate molecule); producing the modified candidate molecule;
determining whether the modified candidate molecule, when produced,
modulates ribosomal activity; and producing the modified candidate
molecule in commercially reasonable amounts for use either alone or
in combination with a pharmaceutically acceptable carrier or
excipient.
[0014] For example, the method optionally comprises the step of,
after synthesizing a candidate molecule, testing whether the
candidate molecule has biological activity, for example, is capable
of modulating ribosome activity in an in vitro assay or is capable
of inhibiting growth of a micro-organism in a growth inhibition
assay. Based on the results of such studies, it is possible to
determine structure-activity-relationships, which can then be used
to design further modifications (for example, the addition,
removal, substitution, or rearrangement of one or more atoms) of
the candidate molecule in order to improve a particular feature of
interest. The modified candidate molecule then can be produced and
assessed for biological activity, as before. These steps can be
repeated one or more times until a modified candidate molecule
having the desired biological activity and pharmacokinetic
properties has been identified. Once such a compound has been
designed, synthesized and tested for activity, it can then be
produced on commercially significant quantities for use as a
pharmaceutical, for example, when formulated with a
pharmaceutically acceptable carrier or excipient.
[0015] In another aspect, the invention provides a computer system
containing information indicative of the three-dimensional
structure of the cyclohexmide molecule when bound to the large
ribosomal subunit. The computer system comprises (a) a memory
having stored therein data indicative of atomic co-ordinates
defining at least a portion of a cycloheximide binding site of a
large subunit of a ribosome and atomic co-ordinates defining at
least a portion of a cycloheximide molecule in association with the
cycloheximide binding site; and (b) a processor in electrical
communication with the memory. The processor comprises a program
for generating a three-dimensional model representative of at least
a portion of the cycloheximide binding site and at least a portion
of the cycloheximide molecule.
[0016] The computer system optionally further comprises a device,
for example, a computer monitor or terminal, for providing a visual
representation of the molecular model. Furthermore, the computer
system optionally further comprises one or more computer programs
for performing rational drug design.
[0017] The molecular model is created from at least a portion of
the atomic co-ordinates recorded on Disk No. 1 under file name
cycloheximide.pdb or from atomic co-ordinates derived by molecular
modeling, for example, homology modeling and/or molecular
replacement, from the atomic co-ordinates recorded on Disk No. 1
under file name cycloheximide.pdb. The cycloheximide binding site
is defined by a plurality of residues set forth in Table 1A or
Table 1B.
[0018] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0020] The objects and features of the invention may be more fully
understood by reference to the drawings described below in
which:
[0021] FIG. 1 is a pictorial representation showing the spatial
relationship of a cycloheximide molecule bound to the large
ribosomal subunit;
[0022] FIG. 2 is a schematic representation of a computer system
useful in molecular modeling a ribosomal subunit and/or for
performing rational drug design; and
[0023] FIG. 3 is a schematic representation of certain potential
drug target sites in a large ribosomal subunit.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0024] As used herein, the term "cycloheximide" refers to a
molecule having the chemical structure I set forth below.
##STR00002##
and pharmaceutically acceptable salts, esters, or prodrugs thereof,
wherein, the numbered atoms in bolded italics represent core atoms.
The core atoms specifically include: C1, N.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.8, C.sub.13, O.sub.14, O.sub.15, C.sub.16,
C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22,
O.sub.23 and O.sub.26.
[0025] As used herein, the term "candidate molecule" refers to
analogs and derivatives of cycloheximide, including molecules that
differ by at least one atom (i.e., contain a least one additional
atom, contain at least one less atom, or contain at least one
substituted atom) from Structure I and that contain (i) at least
eight, more preferably ten of the core atoms in Structure I and/or
(ii) a plurality of atoms disposed within about 5.8 .ANG. of at
least three, more preferably six, more preferably eight of the
residues that define the cycloheximide binding site, when the
candidate molecule is disposed within the 50S ribosomal subunit. It
should be understood that in the candidate molecules, the
connectivity of core atoms can vary. A nonlimiting example of a
candidate molecule might include a structure in which core atoms
such as C.sub.17 and C.sub.18, are not directly bonded to each
other. Candidate molecules also include their pharmaceutically
acceptable salts, esters and prodrugs thereof.
[0026] As used herein, the term "cycloheximide binding site" refers
to a portion of the large ribosomal subunit that interacts or
associates specifically with a cycloheximide molecule.
[0027] As used herein and in reference to a ribosome or ribosomal
subunit, the terms "a portion of" or "a portion of" the
three-dimensional structure of are understood to mean a portion of
the three-dimensional structure of a ribosome or ribosomal subunit,
including charge distribution and hydrophilicity/hydrophobicity
characteristics, formed by at least three, more preferably at least
three to ten, and most preferably at least ten amino acid residues
and/or nucleotide residues of the ribosome or ribosomal subunit.
The residues forming such a portion may be, for example, (i)
contiguous residues based upon, for example, a primary sequence of
a ribosomal RNA or ribosomal protein, (ii) residues which form a
contiguous portion of the three-dimensional structure of the
ribosome or ribosomal subunit, or (c) a combination thereof. As
used herein and in reference to cycloheximide, the terms "a portion
of" or "a portion of the three-dimensional structure of" are
understood to mean a portion of the three-dimensional structure of
cycloheximide, including charge distribution and
hydrophilicity/hydrophobicity characteristics, formed by at least
8, more preferably at least 12 of the atoms included in Structure
I. The atoms forming such a portion may be, for example, (i)
solvent inaccessible atoms buried within the core of the
antibiotic, (ii) solvent accessible atoms of the antibiotic, or
(iii) a combination thereof.
[0028] The structures of two ribosomes, ribosomal subunits or
portions thereof are considered to be the same if they satisfy one
of the following two tests. In a first test, the structures are
considered to be the same if a set of atomic co-ordinates for a
ribosome, ribosomal subunit, or a portion of a large ribosomal
subunit from any source has a root mean square (r.m.s.) deviation
(as determined by MIDAS Plus) of non-hydrogen atoms of less than
about 2.0 .ANG., or more preferably less than about 0.75 .ANG.,
when superimposed on the corresponding non-hydrogen atom positions
of the atomic co-ordinates recorded on compact disk, Disk No. 1,
under file name 1JJ2.pdb. In a second test, the structures are
considered to be the same if the r.m.s. deviation between a set of
atoms in a test structure and a corresponding set of atoms in a
reference structure is less than 2.0 .ANG.. For the purposes of
this test, the set of atoms in the reference structure comprises
(i) at least five of the series of 23S rRNA residues listed below
as 631-633, 835-841, 844-846, 882-885, 1836-1839, 2095-2105,
2474-2478, 2485-2490, 2528-2530, 2532-2543, 2607-2612, 2614-2623,
2642-2648 of the residues of the large ribosomal structure recorded
on compact disk, disk No. 1, under file name 1 JJ2.pdb or (ii) at
least five atoms of residues of the large ribosomal subunit
structure recorded on compact disk, Disk No. 1, under file name
cycloheximide.pdb that are located within 5.8 .ANG. (atom center to
atom center) of the atoms defining the cycloheximide structure
recorded on compact disk, Disk No. 1, under file name
cycloheximide.pdb. The residues in the test structure corresponding
to the ones listed above are identified by sequence alignment using
the program Lasergene v. 5.0 (DNA Star, Inc., Madison, Wis.) with
the default settings. For example, the computer program is used to
align those residues listed above in the Haloarcula marismortui 23S
rRNA sequence with those in the test organism's rRNA. Once aligned,
the corresponding residues in the test organism's rRNA are
identified. The atomic co-ordinates of backbone atoms (P, C5', O5',
C4', C3', O3') of atoms in the test structure are superimposed upon
the corresponding backbone atoms (P, C5', O5', C4', C3', O3') of
the reference structure using the program MIDAS Plus (Ferrin et al.
(1988) J. Mol. Graphics 6: 13-27 and 36-37). The test and reference
structures are considered the same if the r.m.s. deviation between
the two sets of atoms after superpositioning is less than 2.0
.ANG., as determined by MIDAS Plus.
[0029] The structures of two cycloheximide antibiotics or portions
thereof are considered to be the same if the r.m.s. deviation
between the atomic co-ordinates of at least six atoms of a test
structure are within 2.0 .ANG., as determined by MIDAS Plus, of the
corresponding atoms set forth in the cycloheximide reference
structure (as included in file name cycloheximide.pdb, recorded on
compact disk, disk no. 1).
[0030] As used herein, the terms "atoms derived from," "atomic
co-ordinates derived from," and "atomic co-ordinates derived by
molecular modeling from" refer to atoms or atomic co-ordinates
derived, either directly or indirectly, from at least a portion of
the atoms recorded on Disk 1. It is understood that atoms or atomic
co-ordinates derived "directly" from the atomic co-ordinates
recorded on Disk 1 refer to the atoms or portions of the atoms
specifically recorded on Disk 1, which are considered to be primary
atoms or atomic co-ordinates. It is understood that atoms or atomic
co-ordinates derived "indirectly" from the atoms recorded on Disk 1
refers to atoms or atomic co-ordinates that are derived from and
thus are derivatives or transforms of the atoms or atomic
co-ordinates recorded on Disk 1. These co-ordinates are considered
to be secondary atoms or atomic co-ordinates. The secondary atoms
or atomic co-ordinates may be generated from the primary atoms or
atomic co-ordinates by using conventional molecular modeling
techniques. By way of a non limiting example, the atomic
co-ordinates for the H. marismortui large ribosomal subunit as
described hereinbelow are considered to be primary co-ordinates,
whereas the atomic co-ordinates of a mammalian large ribosomal
subunit which are derived from H. marismortui atomic co-ordinates
by molecular modeling, including, for example, homology modeling
and/or molecular replacement, are considered to be secondary
co-ordinates. Both types of atoms and atomic co-ordinates are
considered to be embraced by the invention.
[0031] As used herein the terms "bind," "binding," or "bound" when
used in reference to the association of molecules or chemical
groups refer to any physical contact or association of two or more
atoms, molecules, or chemical groups (e.g., the binding of an
antibiotic with a ribosomal subunit refers to the physical contact
between the antibiotic and the ribosomal subunit). Such contacts
and associations include covalent and non-covalent types of
interactions.
[0032] As used herein, the terms "atomic co-ordinates" or
"structure co-ordinates" refer to mathematical co-ordinates
(represented as "X," "Y" and "Z" values) that describe the
positions of atoms in a crystal of a molecule of interest. The
diffraction data obtained from the crystals are used to calculate
an electron density map of the repeating unit of the crystal. The
electron density maps are used to establish the positions of
individual atoms in the molecule of interest. Those of skill in the
art understand that a set of structure co-ordinates determined by
X-ray crystallography is not without standard error.
[0033] With regard to the list of atomic co-ordinates recorded on
Disk 1, the terms "atomic co-ordinate" or "structure co-ordinates"
refer to the measured position of an atom in the structure in
Protein Data Bank (PDB) format, including X, Y, Z and B, for each.
The term "atom type" refers to the element whose co-ordinates are
measured. The first letter in the column defines the element. The
term "X", "Y", "Z" refers to the crystallographically defined
atomic position of the element measured with respect to the chosen
crystallographic origin. The term "B" refers to a thermal factor
that measures the mean variation of an atom's position with respect
to its average position. The atomic co-ordinates recorded on Disk
1, are recorded in PDB format. For purposes of exemplification
only, in the file 1JJ2.pdb recorded on Disk 1, the "X", "Y", and
"Z" values for atom 1 (an oxygen atom) include "16.071", "148.494",
and "104.415", respectively and the thermal factor for atom 1 is
"83.83".
[0034] Reference is made to the sets of atomic co-ordinates and
related tables included with this specification and submitted on
compact disk (two total compact disks including one original
compact disk, and a duplicate copy of the original compact disk).
As will be apparent to those of ordinary skill in the art, the
atomic structures presented herein are independent of their
orientation, and that the atomic co-ordinates identified herein
merely represent one possible orientation of the molecule of
interest. It is apparent, therefore, that the atomic co-ordinates
identified herein may be mathematically rotated, translated,
scaled, or a combination thereof, without changing the relative
positions of atoms or features of the respective structure. Such
mathematical manipulations are intended to be embraced herein.
[0035] As used herein, the term "A-site" refers to the locus
occupied by an aminoacyl-tRNA molecule immediately prior to its
participation in the peptide-bond forming reaction.
[0036] As used herein, the term "E-site" refers to the locus
occupied by a deacylated tRNA molecule as it leaves the ribosome
following its participation in peptide-bond formation.
[0037] As used herein, the term "P-site" refers to the locus
occupied by a peptidyl-tRNA at the time it participates in the
peptide-bond forming reaction.
[0038] As used herein, the term "pharmaceutically acceptable salt,
ester or prodrug thereof" refers to salt, ester, or prodrug
derivatives of the compound of interest. By "pharmaceutically
acceptable" is meant a salt, ester, or prodrug of the compound of
interest that is generally intended as a safe and nontoxic
modification as understood by one of ordinary skill in the art. One
of ordinary skill in the art can generally prepare a
pharmaceutically acceptable salt, ester or prodrug of a compound of
interest using standard chemical techniques. A pharmaceutically
acceptable salt is where the compound of interest is modified by
making an acid or base salt thereof. A pharmaceutically acceptable
ester is where a hydroxyl group of the compound of interest, if
present, is esterified to make an ester, i.e., typically a simple
ester such as a methyl or ethyl ester or the like. A
pharmaceutically acceptable prodrug is where a functional group of
the compound of interest is modified, typically to alter the
physical characteristics of the compound of interest for
formulation, drug delivery, or other such purposes. An example of a
prodrug would be an acetamide derivative of an amine containing
drug.
[0039] As used herein, the term "ribofunctional locus" refers to a
region of the ribosome or ribosomal subunit that participates,
either actively or passively, in protein or polypeptide synthesis
within the ribosome or ribosomal subunit and/or export or
translocation of a protein or polypeptide out of a ribosome. The
ribofunctional locus can include, for example, a portion of a
peptidyl transferase site, an A-site, a P-site, an E-site, an
elongation factor binding domain, a polypeptide exit tunnel, and a
signal recognition particle (SRP) binding domain. It is understood
that the ribofunctional locus will not only have a certain topology
but also a particular surface chemistry defined by atoms that, for
example, participate in hydrogen bonding (for example, proton
donors and/or acceptors), have specific electrostatic properties
and/or hydrophilic or hydrophobic character. It is understood that
certain antibiotics, for example, cycloheximide, bind to at least a
portion of a ribofunctional locus, for example, a least a portion
of the E-site.
[0040] As used herein, the term "ribosomal subunit" refers to one
of the two subunits of the ribosome that can function independently
during the initiation phase of protein synthesis but which both
together constitute a ribosome. For example, a prokaryotic ribosome
comprises a 50S subunit (large subunit) and a 30S subunit (small
subunit).
II. Structure of Cycloheximide in Association with a Large
Ribosomal Subunit and Uses Thereof
[0041] A. Three-Dimensional Structure of Cycloheximide
[0042] The present invention is based, in part, upon the
three-dimensional structure of cycloheximide when associated with
the large ribosomal subunit. Crystals of the H. marismortui large
ribosomal subunit were soaked with cycloheximide at a concentration
of about 30 mM, which is equivalent to about 0.008% (w/w), and the
structure of the large ribosome subunit complexed with
cycloheximide was resolved via X-ray diffraction studies.
[0043] Briefly, a small amount of a concentrated cycloheximide
solution was added to a large subunit crystal suspended in
stabilization solution and incubated for several hours. Following
freezing and the other procedures normally used to prepare such
crystals for experimental use, X-ray diffraction data were
collected from the cycloheximide containing crystals. Because the
crystals were isomorphous with crystals already described (see, for
example, U.S. Patent Application Publication Nos. US 2002/0086308
A1, published Jul. 4, 2002, US 2003/0153002, published Aug. 14,
2003 and US 2003/0171327, published Sep. 11, 2003), the phases
obtained for native crystal were combined with the diffraction
intensities obtained from the cycloheximide-soaked crystal to
obtain a structure for the latter. The position of the
cycloheximide in the crystal was revealed most clearly in
difference electron density maps, which are electron density maps
computed using the phases just referred to and amplitudes obtained
by subtracting the amplitudes of crystals not containing
cycloheximide from the (suitably scaled) amplitudes of those that
contain cycloheximide. By using the foregoing methods, it was
possible to determine the atomic co-ordinates that show the spatial
relationship between the cycloheximide and its binding site within
the large ribosomal subunit.
[0044] The atomic co-ordinates of the large ribosomal subunit
associated with cycloheximide are recorded on compact disk, Disk
No. 1, under file name cycloheximide.pdb. The residues that, either
alone or in combination, define the cycloheximide binding site are
identified in Table 1. In one embodiment, the cycloheximide binding
site includes, without limitation, a plurality (e.g., at least
three) of the residues (including, for example, one or more of
residues 2431, 2459, and 2460) listed in Table 1A. In addition,
FIG. 1 shows the spatial relationship between cycloheximide and the
large ribosomal subunit, when the cycloheximide is disposed in, or
otherwise associated with the large ribosomal subunit. FIG. 1 shows
cycloheximide bound specifically to the cycloheximide binding site.
Under the conditions of this experiment, cycloheximide bound to a
single location in the large ribosomal subunit and, therefore, did
not bind randomly, i.e. non-specifically, to multiple other sites
of the large ribosomal subunit.
[0045] Based on these studies and published work from other
laboratories, e.g. Schmeing et al., RNA, 9, 1345-52 (2003), it
appears that cycloheximide binds in a pocket that is normally
occupied by A76 of a deacylated tRNA. Without wishing to be bound
by theory, this observation suggests that cycloheximide prevents
the normal binding of a deacylated E-site tRNA.
[0046] Based on these studies, there are differences between the
Haloarcula marismortui cycloheximide binding site and the
homologous prokaryotic bacterial counterpart binding site as
defined in Table 1. Prokaryotic bacterial 50S subunits have a
2-residue RNA insertion, namely residues U2431 and A2432 in E.
coli, while eukaryotic and archea bacterial 50S subunits have a
protein, L44E, that is lacking in prokaryotes. For example, the
design of anti-bacterials based on the structure of cycloheximide
bound to the 50S subunit can be pursued by taking these differences
into account.
[0047] B. Rational Drug Design
[0048] 1. Introduction
[0049] The atomic co-ordinates defining the three-dimensional
structure of at least a portion of cycloheximide, when associated
with a large ribosomal subunit, can be used in rational drug design
(RDD) to design a novel molecule of interest, for example, a novel
modulator (for example, inducer, mimetic or inhibitor) of ribosome
function. Furthermore, it is contemplated that, by using the
principles disclosed herein, the skilled artisan can design, make,
test, refine and use analogs specifically engineered to reduce,
disrupt, or otherwise or inhibit ribosomal function in an organism
or species of interest. For example, by using the principles
discussed herein, the skilled artisan can engineer new molecules
that specifically target and inhibit ribosomal function in a
pathogen, for example, a particular prokaryotic organism, while
preserving ribosomal function in a host, for example, a eukaryotic
organism, specifically a mammal, and more specifically, a human. As
a result, the atomic co-ordinates provided herein permit the
skilled artisan to design new antibiotics that can kill certain
pathogenic organisms while having little or no toxicity in the
intended recipient, for example, a human.
[0050] It is contemplated that RDD using atomic co-ordinates of the
large ribosomal subunit can be facilitated most readily via
computer-assisted drug design (CADD) using conventional computer
hardware and software known in the art. The candidate molecules may
be designed de novo or may be designed as a modified version of an
already existing molecule, for example, a pre-existing antibiotic
or protein synthesis modulator, using conventional methodologies.
Once designed, candidate molecules can be synthesized using
standard methodologies known in the art. Following synthesis, the
candidate molecules can be screened for bioactivity, for example,
their ability to interact with or bind specifically to a ribosome
or a ribosomal subunit, or by their ability to reduce or inhibit
ribosome function. Based in part upon these results, the candidate
molecules can be refined iteratively using one or more of the
foregoing steps to produce a more desirable molecule with a desired
biological activity. The processes can be repeated as many times as
necessary to obtain molecules with desirable binding properties
and/or biological activities. The resulting molecules can be useful
in treating, inhibiting or preventing the biological activities of
target organisms, thereby killing the organism or impeding its
growth. Alternatively, the resulting molecules can be useful for
treating, inhibiting or preventing microbial infections in any
organism, particularly animals, more particularly humans.
[0051] 2. Identification of Candidate Molecules
[0052] It is contemplated that the design of candidate molecules of
interest can be facilitated by conventional ball and stick-type
modeling procedures. However, in view of the size and complexity of
the molecules of interest when associated with a large ribosomal
subunit, it is contemplated that the ability to design candidate
molecules can be enhanced significantly using computer-based
modeling and design protocols.
[0053] a. Molecular Modeling
[0054] It is contemplated that the design of candidate molecules,
as discussed in detail hereinbelow, can be facilitated using
conventional computers or workstations, available commercially
from, for example, Silicon Graphics Inc. and Sun Microsystems, Inc.
running, for example, UNIX based, Windows NT on IBM OS/2 operating
systems, and capable of running conventional computer programs for
molecular modeling and rational drug design.
[0055] It is understood that any computer system having the overall
characteristics set forth in FIG. 2 can be useful in the practice
of the invention. More specifically, FIG. 2, is a schematic
representation of a typical computer work station having in
electrical communication (100) with one another via, for example,
an internal bus or external network, a central processing unit
(101), a random access memory (RAM) (102), a read only memory (ROM)
(103), a monitor or terminal (104), and optionally an external
storage device, for example, a diskette, CD ROM, or magnetic tape
(105).
[0056] The terms "computer system" or "computer-based system" refer
to hardware, software, and data storage devices capable of, for
example, storing, analyzing, manipulating and/or presenting the
atomic co-ordinates of the invention. The data storage device can
be any memory device which can store data indicative of atomic
co-ordinates.
[0057] The data indicative of atomic co-ordinates can be recorded
on computer readable medium including, for example, any medium
which can be read and accessed directly by a computer. Such media
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc storage medium, and magnetic tape; optical
storage media such as optical discs or CD-ROMs; electrical storage
media such as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable media
can be used to create a manufacture comprising computer readable
medium having recorded thereon data indicative of the atomic
co-ordinates of the invention. A skilled artisan can readily adopt
any of the presently known methods for recording information on
computer readable medium to generate manufactures comprising data
indicative of atomic co-ordinates of the invention.
[0058] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon data indicative of atomic co-ordinates of the
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the atomic co-ordinates of the present invention on
computer readable medium. The foregoing data can be represented in
a word processing text file, formatted in commercially-available
software such as WordPerfect and MICROSOFT Word, or represented in
the form of an ASCII file, stored in a database application, such
as DB2, Sybase, Oracle, or the like. A skilled artisan can readily
adapt any number of data processor structuring formats (e.g. text
file or database) in order to obtain computer readable medium
having recorded thereon the information of the present
invention.
[0059] By providing a computer readable medium having stored
therein data indicative of atomic co-ordinates, a skilled artisan
can routinely access the data to model the protein synthesis
modulator of interest or derivatives or analogs thereof, either
alone or in combination with a ribosome or ribosomal subunit.
Computer algorithms are publicly and commercially available which
allow a skilled artisan to access this data provided in a computer
readable medium and analyze it for molecular modeling and/or RDD.
See, e.g., Biotechnology Software Directory, MaryAnn Liebert Publ.,
New York, N.Y. (1995).
[0060] Although computers are not required, molecular modeling can
be most readily facilitated by using computers to build realistic
models of the protein synthesis modulator or analogs thereof,
either or alone or in combination with a ribosome or ribosomal
subunit. The methods utilized in molecular modeling range from
molecular graphics (i.e., three-dimensional representations) to
computational chemistry (i.e., calculations of the physical and
chemical properties) to make predictions about the binding of the
smaller molecules or their activities; to design new molecules; and
to predict novel molecules, including ligands such as drugs, for
chemical synthesis.
[0061] For basic information on molecular modeling, see, for
example, M. Schlecht, Molecular Modeling on the PC (1998) John
Wiley & Sons; Gans et al., Fundamental Principals of Molecular
Modeling (1996) Plenum Pub. Corp.; N. C. Cohen, ed., Guidebook on
Molecular Modeling in Drug Design (1996) Academic Press; and W. B.
Smith, Introduction to Theoretical Organic Chemistry and Molecular
Modeling (1996). U.S. patents which provide detailed information on
molecular modeling include, for example: U.S. Pat. Nos. 6,093,573;
6,080,576; 6,075,014; 6,075,123; 6,071,700; 5,994,503; 5,884,230;
5,612,894; 5,583,973; 5,030,103; 4,906,122; and 4,812,128.
[0062] Three-dimensional modeling can include, but is not limited
to, making three-dimensional representations of structures, drawing
pictures of structures, building physical models of structures, and
determining the structures of antibiotics, protein synthesis
modulators, and/or derivatives or analogs thereof, ribosomes,
ribosomal subunits, and complexes thereof. The appropriate
co-ordinates can be entered into one or more computer programs for
molecular modeling, as known in the art. By way of illustration, a
list of computer programs useful for viewing or manipulating
three-dimensional structures include: Midas (University of
California, San Francisco); MidasPlus (University of California,
San Francisco); MOIL (University of Illinois); Yummie (Yale
University); Sybyl (Tripos, Inc.); Insight/Discover (Biosym
Technologies); MacroModel (Columbia University); Quanta (Molecular
Simulations, Inc.); Cerius (Molecular Simulations, Inc.); Alchemy
(Tripos, Inc.); LabVision (Tripos, Inc.); Rasmol (Glaxo Research
and Development); Ribbon (University of Alabama); NAOMI (Oxford
University); Explorer Eyechem (Silicon Graphics, Inc.); Univision
(Cray Research); Molscript (Uppsala University); Chem-3D (Cambridge
Scientific); Chain (Baylor College of Medicine); O (Uppsala
University); GRASP (Columbia University); X-Plor (Molecular
Simulations, Inc.; Yale University); Spartan (Wavefunction, Inc.);
Catalyst (Molecular Simulations, Inc.); Molcadd (Tripos, Inc.); VMD
(University of Illinois/Beckman Institute); Sculpt (Interactive
Simulations, Inc.); Procheck (Brookhaven National Library); DGEOM
(QCPE); RE_VIEW (Brunell University); Modeller (Birbeck College,
University of London); Xmol (Minnesota Supercomputing Center);
Protein Expert (Cambridge Scientific); HyperChem (Hypercube); MD
Display (University of Washington); PKB (National Center for
Biotechnology Information, NIH); ChemX (Chemical Design, Ltd.);
Cameleon (Oxford Molecular, Inc.); and Iditis (Oxford Molecular,
Inc.).
[0063] One approach to RDD is to search for known molecular
structures that might bind to a site of interest. Using molecular
modeling, RDD programs can look at a range of different molecular
structures of molecules that can fit into a site of interest, and
by moving them on the computer screen or via computation it can be
decided which structures actually fit the site well (William Bains
(1998) Biotechnology from A to Z, second edition, Oxford University
Press, p. 259).
[0064] An alternative but related approach starts with the known
structure of a complex with a small molecule ligand and models
modifications of that small molecule in an effort to make
additional favorable interactions with a ribosome or ribosomal
subunit.
[0065] The present invention permits the use of molecular and
computer modeling techniques to design and select novel molecules,
such as antibiotics or other therapeutic agents, that interact with
ribosomes and ribosomal subunits. Such antibiotics and other types
of therapeutic agents include, but are not limited to, antifungals,
antivirals, antibacterials, insecticides, herbicides, miticides,
rodentcides, etc.
[0066] In order to facilitate molecular modeling and/or RDD the
skilled artisan can use some or all of the atomic co-ordinates
recorded on Disk 1, under file names cycloheximide.pdb and
1JJ2.pdb. By using the foregoing atomic co-ordinates, the skilled
artisan can design new modulators of protein synthesis that can be
tailored to be effective against ribosomes from one or more species
but which have little or no effect on ribosomes of other species.
The atomic co-ordinates provided herein also permit probing the
three-dimensional structure of a ribosome or ribosome subunit or a
portion thereof with molecules composed of a variety of different
chemical features to determine optimal sites for interaction
between candidate molecules and the ribosome or ribosomal subunit.
Small molecules that bind those sites can then be designed,
synthesized and tested for inhibitory activity (Travis, J. (1993)
Science 262: 1374). These molecules can represent lead compounds
from which further drug-like compounds can be synthesized.
[0067] b. Identification of Target Sites
[0068] The atomic co-ordinates of the invention permit the skilled
artisan to identify target locations in a ribosome or large
ribosomal subunit that can serve as a starting point in rational
drug design. As a threshold matter, the atomic co-ordinates permit
the skilled artisan to identify specific regions within a ribosome
or ribosomal subunit that are involved with protein synthesis in,
and/or protein secretion out of, the ribosome. Furthermore, the
atomic co-ordinates permit a skilled artisan to further identify
portions of these regions that are conserved or are not conserved
between different organisms. For example, by identifying portions
of these regions that are conserved among certain pathogens, for
example, certain prokaryotes, but are not conserved in a host
organism, for example, a eukaryote, more preferably a mammal, the
skilled artisan can design molecules that selectively inhibit or
disrupt protein synthesis activity of the pathogen's but not the
host's ribosomes. Furthermore, by analyzing regions that are either
conserved or non-conserved between certain pathogens, it can be
possible to design broad or narrow spectrum protein synthesis
inhibitors, e.g., antibiotics, as a particular necessity
arises.
[0069] FIG. 3 is a schematic representation of a large ribosomal
subunit that identifies a variety of exemplary target sites that
appear to participate in protein synthesis within the ribosome
and/or the export or translocation of the newly synthesized protein
out of the ribosome. Inspection of the atomic co-ordinates of the
H. marismortui 50S ribosomal subunit has identified a variety of
target regions that can serve as a basis for the rational drug
design of new or modified protein synthesis inhibitors. The target
regions include a P-site (200 in FIG. 3), an A-site (201 in FIG.
3), a peptidyl transferase site (202 in FIG. 3) which includes a
portion of the P-site and the A-site, an E-site (203 in FIG. 3), a
factor binding domain (204 in FIG. 3) including, for example, the
EF-Tu binding domain and the EF-G binding domain, the polypeptide
exit tunnel (205 in FIG. 3) including cavities defined by the wall
of the exit tunnel, and the signal recognition particle binding
domain (206 in FIG. 3). The specific locations of certain of these
target regions within the large ribosomal subunit are described,
for example, in U.S. Patent Application Publication Nos. US
2002/0086308 and US 2003/0153002. Cyclohexmide appears to bind the
ribosome at the E-site (203 in FIG. 3).
[0070] Table 1A identifies the residues in the H. marismortui 50S
ribosomal subunit that together define at least a portion of a
cycloheximide binding site (5.8 .ANG. shell). In addition, Table 1A
identifies the corresponding residues that define at least a
portion of the cycloheximide binding site in E. coli, Rattus,
human, and human mitochondria large subunit. Table 1B identifies
the residues in the H. marismortui 50S ribosomal subunit that
together define a broader portion of a cycloheximide binding site
(5.8 .ANG.-12.6 .ANG. shell). The conserved and non-conserved
residues were identified by comparison of sequences from the
structure of H. marismortui rRNA or ribosomal protein that form the
cycloheximide binding site with the corresponding sequences of
aligned genomic DNA encoding either the homologous rRNA or
ribosomal protein from the other organisms. Sequence alignments can
be determined with the program MegAlign (DNASTAR, Madison, Wis.,
USA) using default parameters.
TABLE-US-00001 TABLE 1A Residues that Define the Cycloheximide
Binding Pocket (5.8 .ANG. shell) Corre- Correspond- Correspond-
Corresponding sponding ing ing Residue in H. marismortui Residue in
Residue in Residue in Human Residue E. coli Rattus Human
Mitochondria 23S RNA G219 G248 G91 G91 G78 C220 C249 C92 C92 C79
G221 G250 G93 G93 G80 A2430 U2393 U4061 U4300 U1228 C2431 C2394
C4062 C4301 C1229 C2432 C2395 C4063 C4302 C1230 G2459 G2421 G4091
G4330 G1239 A2460 C2422 G4092 G4331 A1240 A2468 A2433 A4100 A4339
C1250 Protein L44E K51 -- T52 T52 -- K54 -- I55 I55 -- P56 -- R57
R57 -- G57 -- K58 K58 -- G58 -- K59 K59 -- Residues were determined
by a 5.8 angstrom distance measurement between the atom centers of
cycloheximide and the 50S ribosome using the program SPOCK.sup.a.
Conserved residues were determined by comparison between the
proposed structures of H. marismortui.sup.b, E. coli.sup.c, Rattus
norvegicus.sup.d, Human.sup.e, and Human Mitochondria.sup.f. A dash
(--) means a homologous residue is not present. .sup.aChristopher,
Jon A. (1998) SPOCK: The Structural Properties Observation and
Calculation Kit (Program Manual), The Center for Macromolecular
Design, Texas A&M University, College Station, TX.
.sup.bGenbank entries AF034620 (RNA) and P32411 (protein L44E). The
sequences of 1JJ21.pdb and Genbank entry AF034620 are identical but
the numbering is offset by one residue for the Genbank entry (i.e.,
lower by one). .sup.cGenbank entry NC_000913 (RNA). .sup.dGenbank
entries X01069 (RNA) and R6RT36 (protein L44E). .sup.eGenbank
entries M11167 (RNA) and P09896 (protein L44E). .sup.fGenbank entry
V00710 (RNA).
TABLE-US-00002 TABLE 1B Residues that Further Define the
Cycloheximide Binding Pocket (5.8 .ANG.-12.6 .ANG. shell) Corre-
Corre- Corresponding sponding Corresponding sponding Residue in H.
marismortui Residue in Residue in Residue in Human Residue E. coil
Rattus Human Mitochondria 23S rRNA A167 A197 G40 G40 C55 C168 C198
C41 C41 C56 A169 A199 A42 A42 A57 U170 U200 U43 U43 U58 C218 G247
G90 G90 G77 G2428 G2391 G4059 G4298 G1226 A2429 A2392 A4060 A4299
A1227 A2433 G2396 U4064 U4303 A1231 A2434 G2397 U4065 U4304 A1232
U2457 U2419 G4089 G4328 U1237 U2458 C2420 A4090 A4329 U1238 U2461
C2424 U4093 U4332 C1242 G2462 A2425 G4094 G4333 A1243 G2466 G2429
G4098 G4337 G1247 A2467 A2430 A4099 A4338 A1248 A2469 A2434 A4101
A4340 A1251 Protein L44E S27 -- K28 K28 -- Q30 -- D31 D31 -- W35 --
Q36 Q36 -- I36 -- G37 G37 -- Q39 -- R40 R40 -- R40 -- Y41 Y41 --
N43 -- K44 K44 -- N48 -- G49 G49 -- D49 -- G50 G50 -- G50 -- Q51
Q51 -- F52 -- K53 K53 -- S53 -- P54 P54 -- V55 -- F56 F56 -- D59 --
A60 A60 -- K60 -- K61 K61 -- P61 -- T62 T62 -- T62 -- T63 T63 --
K63 -- K64 K64 -- K64 -- K65 K65 -- D66 -- V67 V67 -- R84 -- R87
R87 -- Protein L15 R35 V46 H40 H40 P63 D36 R47 H41 H41 R64 P45 P56
P50 P50 P73 L46 L61 F53 F53 I78 G47 P62 G54 G54 P79 K48 K63 K55 K55
K80 Residues were determined as a 5.8-12.6 angstrom distance
measurement between the atom centers of cycloheximide and the 50S
ribosome using the program SPOCK.sup.a. Conserved residues were
determined by comparison between the proposed structures of H.
marismortui.sup.b, E. coli.sup.c, and Rattus norvegicus.sup.d,
Human.sup.e, and Human Mitochondria.sup.f. A dash (--) means a
homologous residue is not present. .sup.aChristopher, Jon A. (1998)
SPOCK: The Structural Properties Observation and Calculation Kit
(Program Manual), The Center for Macromolecular Design, Texas
A&M University, College Station, TX. .sup.bGenbank entries
AF034620 (RNA), P32411 and R6HS15 (proteins L44E and L15,
respectively). The sequences of 1JJ21.pdb and Genbank entry
AF034620 are identical but the numbering is offset by one residue
for the Genbank entry (i.e.lower by one). .sup.cGenbank entries
NC_000913 (RNA) and NP_417760 (protein L15). .sup.dGenbank entries
X01069 (RNA), R6RT36 and R5RTLA (proteins L44E and L15,
respectively). .sup.eGenbank entries M11167 (RNA), P09896 and
NP_000981 (proteins L44E and L15, respectively). .sup.fGenbank
entries V00710 (RNA) and NP_054894 (protein L15).
[0071] The skilled artisan, when in possession of the foregoing or
other exemplary target sites, can use the process of rational drug
design to identify molecules that potentially bind to one or more
of the target sites and/or inhibit ribosomal activity. Furthermore,
by taking into account which of the residues that define the target
site are conserved between pathogens but not conserved between host
species, the skilled artisan can design new species-specific
protein synthesis inhibitors. It is apparent that the skilled
artisan can take advantage of the regions that are not conserved
between E. coli and rat or human to provide target regions for
rational drug design.
[0072] c. Identification of Candidate Molecules
[0073] It is contemplated that candidate molecules that modulate
protein biosynthesis can be designed entirely de novo or can be
based upon one or more pre-existing molecules. Either of these
approaches can be facilitated by computationally screening
databases and libraries of small molecules for chemical entities,
agents, ligands, or compounds that can bind in whole, or in part,
to ribosomes and ribosomal subunits, more preferably to large
ribosomal subunits, and even more preferably to 50S ribosomal
subunits. In this screening, the quality of fit of such entities or
compounds to the binding site or sites can be judged either by
shape complementarity or by estimated interaction energy (Meng et
al. (1992) J. Comp. Chem. 13: 505-524).
[0074] The design of molecules that bind to and/or inhibit the
functional activity of ribosomes or ribosomal subunits generally
involves consideration of two factors. First, the molecule must be
capable of physically and structurally associating with the large
ribosomal subunit. Non-covalent molecular interactions important in
the association of ribosomes and ribosomal subunits with the
molecule, include hydrogen bonding, van der Waals and hydrophobic
interactions. Second, the molecule must be able to assume a
conformation that allows it to associate with the ribosomes or
ribosomal subunits, and more preferably with the large ribosomal
subunits.
[0075] The candidate molecule and analogs or derivatives of the
candidate molecule (also referred to as a modified candidate
molecule) preferably bind specifically or have binding specificity
to the large ribosomal unit, as does cycloheximide. For example,
the candidate molecule and the modified candidate molecule in order
to impart their biological effect on ribosome function preferably
bind to, for example, no greater than 5, optionally less than 5,
optionally less than 4, optionally less than 3, and optionally less
than 2 sites in a large ribosomal subunit.
[0076] Preferably, the candidate molecule or modified candidate
molecule binds to, for example, at least a portion of the
corresponding cycloheximide binding site of the large ribosomal
subunit, to modulate ribosome function. As discussed hereinabove,
the cycloheximide binding site comprises a plurality of residues
listed in Table 1. Table 1A lists, for example, 14 residues that
contribute to the cycloheximide binding pocket. The candidate
molecule and the modified candidate molecule when they bind
specifically to the cycloheximide binding site contact 4 to 14, 5
to 13, 6 to 12, 7 to 11, and 8 to 10 of the residues listed in
Table 1A. Preferably, when the candidate molecule and the modified
candidate molecule binds specifically to the cycloheximide binding
site, the molecule contacts at least 6, optionally at least 8, and
optionally at least 10, of the residues listed in Table 1A.
[0077] Although certain portions of the candidate molecule not
directly participate in this association with a ribosome or
ribosomal subunits, those portions may still influence the overall
conformation of the molecule. This, in turn, may have an effect on
binding affinities, therapeutic efficacy, drug-like qualities, and
potency. Such conformational requirements include the overall
three-dimensional structure and orientation of the chemical entity
or molecule in relation to all or a portion of the active site or
other region of the ribosomes or ribosomal subunits, or the spacing
between functional groups of a molecule comprising several chemical
entities that directly interact with the ribosomes or ribosomal
subunits, more preferably with the large ribosomal subunits, and
even more preferably with the 50S ribosomal subunit.
[0078] The potential, predicted, inhibitory or binding effect of a
molecule on ribosomes and ribosomal subunits can be analyzed prior
to its actual synthesis and testing by the use of computer modeling
techniques. If the theoretical structure of the given molecule
suggests insufficient interaction and association between it and
ribosomes or ribosomal subunits, synthesis and testing of the
molecule is obviated. However, if computer modeling indicates a
strong interaction (for example, a binding interaction similar to
that of a native cycloheximide molecule), the molecule can then be
synthesized and tested for its ability to interact with the
ribosomes or ribosomal subunits and inhibit protein synthesis. In
this manner, synthesis of potentially inoperative molecules can be
avoided. The synthesis and characterization of such molecules
permits the development of structure-activity relationships, which
can then be used in the iterative refinement of the molecules of
interest.
[0079] d. De Novo Design
[0080] One skilled in the art can use one of several methods to
identify chemical moieties or entities, compounds, or other agents
for their ability to associate with a preselected target site
within a ribosomes or ribosomal subunit. This process can begin by
visual inspection or computer assisted modeling of, for example,
the target site on the computer screen based on the atomic
co-ordinates of the antibiotic and/or protein synthesis modulator
either alone or in association with the 50S ribosomal subunit. In
one embodiment, molecule design uses computer modeling programs
which calculate how different molecules interact with the various
sites of the ribosome, ribosomal subunit, or a fragment thereof.
Selected chemical moieties or entities, compounds, or agents can
then be positioned in a variety of orientations, or docked, within
at least a portion of the target site of a ribosome or ribosomal
subunit, more preferably of a large ribosomal subunit, and even
more preferably of a 50S ribosomal subunit. Databases of chemical
structures are available from, for example, Cambridge
Crystallographic Data Center (Cambridge, U.K.) and Chemical
Abstracts Service (Columbus, Ohio). Docking can be accomplished
using software such as Cerius, Quanta or Sybyl, followed by energy
minimization and molecular dynamics with standard molecular
mechanics forcefields, such as OPLS-AA, CHARMM or AMBER.
[0081] Specialized computer programs can also assist in the process
of selecting chemical entities. These include, but are not limited
to: [0082] (1) GRID (Goodford, P. J., "A Computational Procedure
for Determining Energetically Favorable Binding Sites on
Biologically Important Macromolecules" (1985) J. Med. Chem. 28,
849-857). Software such as GRID, a program that determines probable
interaction sites between probes with various functional group
characteristics and the macromolecular surface, can be used to
analyze the surface sites to determine structures of similar
inhibiting proteins or molecules. The GRID calculations, with
suitable inhibiting groups on molecules (e.g., protonated primary
amines) as the probe, are used to identify potential hotspots
around accessible positions at suitable energy contour levels. GRID
is available from Oxford University, Oxford, UK. [0083] (2) MCSS
(Miranker, A. and M. Karplus (1991) "Functionality Maps of Binding
Sites: A Multiple Copy Simultaneous Search Method." Proteins:
Structure, Function and Genetics 11: 29-34). MCSS is available from
Molecular Simulations, Burlington, Mass. [0084] (3) AUTODOCK
(Goodsell, D. S, and A. J. Olsen (1990) "Automated Docking of
Substrates to Proteins by Simulated Annealing" Proteins: Structure,
Function, and Genetics 8: 195-202). AUTODOCK is available from
Scripps Research Institute, La Jolla, Calif. [0085] (4) DOCK
(Kuntz, I. D. et al. (1982) "A Geometric Approach to
Macromolecule-Ligand Interactions" J. Mol. Biol. 161: 269-288). The
program DOCK can be used to analyze an active site or ligand
binding site and suggest ligands with complementary steric
properties. DOCK is available from University of California, San
Francisco, Calif. [0086] (5) ALADDIN (Van Drie et al. (1989)
"ALADDIN: An Integrated Tool of Computer Assisted Molecular Design
and Pharmacophore Recognition From Geometric, Steric and
Substructure Searching of Three-Dimensional Structures" J.
Comp-Aided Mol. Des. 3: 225). [0087] (6) CLIX (Davie and Lawrence
(1992) "CLIX: A Search Algorithm for Funding Novel Ligands Capable
of Binding Proteins of Known Three-Dimensional Structure" Proteins
12: 31-41). [0088] (7) GROUPBUILD (Rotstein and Murcko (1993)
"GroupBuild: A Fragment-Based Method for De Novo Drug Design" J.
Med. Chem 36: 1700). [0089] (8) GROW (Moon and Howe (1991)
"Computer Design of Bioactive Molecules: A Method for
Receptor-Based De Novo Ligand Design" Proteins 11: 314).
[0090] Once suitable chemical moieties or entities, compounds, or
agents have been selected, they can be assembled into a single
molecule. Assembly can proceed by visual inspection and/or computer
modeling and computational analysis of the spatial relationship of
the chemical moieties or entities, compounds or agents with respect
to one another in three-dimensional space. This could then be
followed by model building using software such as Quanta or
Sybyl.
[0091] Useful programs to aid one of skill in the art in connecting
the individual chemical entities, compounds, or agents include but
are not limited to: [0092] (1) CAVEAT (Bartlett, P. A. et al.
(1989) "CAVEAT: A Program to Facilitate the Structure-Derived
Design of Biologically Active Molecules". In molecular Recognition
in Chemical and Biological Problems", Special Pub., Royal Chem.
Soc. 78: 82-196) and (Bacon et al. (1992) J. Mol. Biol. 225:
849-858). CAVEAT uses databases of cyclic compounds which can act
as "spacers" to connect any number of chemical fragments already
positioned in the active site. This allows one skilled in the art
to quickly generate hundreds of possible ways to connect the
fragments already known or suspected to be necessary for tight
binding. CAVEAT is available from the University of California,
Berkeley, Calif. [0093] (2) 3D Database systems such as MACCS-3D
(MDL Information Systems, San Leandro, (CA). This area is reviewed
in Martin, Y. C., (1992) "3D Database Searching in Drug Design", J.
Med. Chem. 35: 2145-2154. [0094] (3) HOOK (available from Molecular
Simulations, Burlington, Mass.).
[0095] Instead of proceeding to build a molecule of interest in a
step-wise fashion one chemical entity at a time as described above,
the molecule of interest can be designed as a whole using either an
empty active site or optionally including some portion or portions
of a preselected molecule. Software that implements these methods
include: [0096] (1) LUDI (Bohm, H.-J. (1992) "The Computer Program
LUDI: A New Method for the De Novo Design of Enzyme Inhibitors" J.
Comp. Aid. Molec. Design 6: 61-78). The program LUDI can determine
a list of interaction sites into which to place both hydrogen
bonding and hydrophobic fragments. LUDI then uses a library of
approximately 600 linkers to connect up to four different
interaction sites into fragments. Then smaller "bridging" groups
such as --CH.sub.2-- and --COO-- are used to connect these
fragments. For example, for the enzyme DHFR, the placements of key
functional groups in the well-known inhibitor methotrexate were
reproduced by LUDI. See also, Rotstein and Murcko, (1992) J. Med.
Chem. 36:1700-1710. LUDI is available from Biosym Technologies, San
Diego, Calif. [0097] (2) LEGEND (Nishibata, Y. and A. Itai (1991)
Tetrahedron 47, 8985) (available from Molecular Simulations,
Burlington, Mass.). [0098] (3) LeapFrog (available from Tripos
Associates, St. Louis, Mo.). [0099] (4) Aladdin (available from
Daylight Chemical Information Systems, Irvine, Calif.)
[0100] Other molecular modeling techniques can also be employed in
accordance with this invention. See, e.g., Cohen, N. C. et al.
(1990) "Molecular Modeling Software and Methods for Medicinal
Chemistry, J. Med. Chem. 33: 883-894. See also, Navia, M. A. and M.
A. Murcko (1992) "The Use of Structural Information in Drug
Design", Current Opinions in Structural Biology 2: 202-210; and
Jorgensen (1998) "BOSS--Biochemical and Organic Simulation System"
in the Encyclopedia of Computational Chemistry (P.V.R. Schleyer,
ed.) Wiley & Sonstra., Athens, U.S.A. 5: 3281-3285).
[0101] It is contemplated that during modeling, it can be possible
to introduce into the molecule of interest, chemical moieties that
can be beneficial for a molecule that is to be administered as a
pharmaceutical. For example, it can be possible to introduce into
or omit from the molecule of interest, chemical moieties that
cannot directly affect binding of the molecule to the target area
but which contribute, for example, to the overall solubility of the
molecule in a pharmaceutically acceptable carrier, the
bioavailability of the molecule and/or the toxicity of the
molecule. Considerations and methods for optimizing the
pharmacology of the molecules of interest can be found, for
example, in "Goodman and Gilman's The Pharmacological Basis of
Therapeutics" Eighth Edition (Goodman, Gilman, Rall, Nies, &
Taylor (eds.)). Pergaman Press (1985); Jorgensen & Duffy (2000)
Bioorg. Med. Chem. Lett. 10: 1155-1158.
[0102] Furthermore, the computer program "Qik Prop" can be used to
provide rapid predictions for physically significant descriptions
and pharmaceutically-relevant properties of an organic molecule of
interest. A `Rule of Five` probability scheme can be used to
estimate oral absorption of the newly synthesized compounds
(Lipinski et al. (1997) Adv. Drug Deliv. Rev. 23:3).
[0103] Programs suitable for pharmacophore selection and design
include: [0104] (1) DISCO (Abbot Laboratories, Abbot Park, Ill.).
[0105] (2) Catalyst (Bio-CAD Corp., Mountain View, Calif.). [0106]
(3) Chem DBS-3D (Chemical Design Ltd., Oxford, U.K.).
[0107] Furthermore, the skilled artisan can use the information
available on how to design suitable therapeutically active and
pharmaceutically useful compounds, and use this information in the
design of new protein synthesis modulators. See, for example,
Lipinski et al. (1997) Ad. Drug Deliv. Reviews 23: 3-25; Van de
Waterbeemd et al. (1996) Quantitative Structure-Activity
Relationships 15: 480-490; and Cruciani et al. (2000) Theochem-J.
Mol. Struct. 503: 17-30.
[0108] The entry of the co-ordinates of the ribosome's or ribosomal
subunit's proteins and RNAs into the computer programs discussed
above facilitates the calculation of most probable structure of the
macromolecule, including overall atomic co-ordinates of a ribosome,
ribosomal subunit or a fragment thereof. These structures can be
combined and refined by additional calculations using such programs
to determine the probable or actual three-dimensional structure of
the ribosome, ribosomal subunit or a fragment thereof, including
potential or actual active or binding sites of ligands.
[0109] e. Modification of Existing Molecules
[0110] Instead of designing molecules of interest entirely de nova
it is contemplated that pre-existing molecules or portions thereof
can be used as a starting point for the design of a new candidate.
It is contemplated that many of the approaches useful for designing
molecules de novo can also be useful for modifying existing
molecules.
[0111] It is contemplated that knowledge of the spatial
relationship between a modulator of protein biosynthesis, for
example, an antibiotic, and its respective binding site within a
ribosome permits the design of modified modulator that can have
better binding properties, for example, higher binding affinity
and/or binding specificity, relative to the molecule from which it
was derived. Alternatively, knowledge of modulator contact sites
within a ribosome permits the synthesis of a new molecule that
contains, for example, a portion of a first molecule (for example,
an antibiotic or an analog or derivative) that binds to the contact
site and another portion that contributes additional
functionality.
[0112] It is contemplated that a variety of modified molecules (for
example, modified protein synthesis modulators) can be designed
using the atomic co-ordinates provided herein. For example, it is
contemplated that by knowing the spatial relationship of one or
more of protein synthesis modulators relative to the large
ribosomal subunit it is possible to generate new molecules. The
atomic co-ordinates of each protein synthesis modulator relative to
the large ribosomal subunit provides information on what portions
of the ribosome or ribosomal subunit and the protein synthesis
modulator contact one another. Accordingly, from this information
the skilled artisan can identify contact locations within the
ribosome that can be used for de novo drug design, as discussed
above, but also can identify portions of an protein synthesis
modulator that can act as a ribosome binding domain.
[0113] Furthermore, the atomic co-ordinates provided herein permit
the skilled artisan to use the information pertaining to identify a
ribosome binding domain and to design other modulators of protein
biosynthesis. For example, with an understanding of the ribosome
contact region and the surrounding environment, the skilled artisan
can provide novel molecules, a portion of which is based upon the
antibiotic binding region (binding domain) and another portion of
which (effector domain) can be designed as a novel space filling
domain that sterically inhibits or disrupts protein biosynthesis
within the ribosome or secretion through the polypeptide exit
tunnel. For example, the skilled artisan can combine a ribosome
binding portion of cycloheximide which binds to the E site with,
for example, a novel chemical moiety, for example, a moiety not
present in antibiotics identified to date, that is bulky enough to
block more effectively the binding of deacylated tRNA to the E
site. However, it is contemplated that the skilled artisan can take
advantage of one or more of the many of the antibiotic contact
regions disclosed herein to design entirely new binding and
effector domains. In addition, it is understood that the large
ribosomal subunits of prokaryotic, but not eukaryotic, origin
contain rRNA residues that sterically hinder the binding of
cycloheximide to the large ribosomal subunit.
[0114] The resulting protein synthesis modulators, for example,
protein synthesis inhibitors, can have a molecular weight no
greater than about 1,500, optionally no greater than about 1,000,
optionally no greater than 750 and, optionally no greater than
about 500. The protein synthesis inhibitors can have a molecular
weight in the range from about 250 to about 1500, and optionally in
the range from about 500 to about 1200. In addition, the protein
synthesis inhibitors have a minimal inhibitor concentration
optionally no greater than 50 .mu.M, optionally no greater than 10
.mu.M, and optionally no greater than 1 .mu.M to inhibit 50%
activity (IC.sub.50) in a biological assay, for example, an in
vitro translation assay, for example, an E. coli translation assay.
The protein synthesis inhibitors can have an IC.sub.50 in the range
from about 0.001 .mu.M to about 50 .mu.M, optionally in the range
from about 0.01 .mu.M to about 10 .mu.M, and optionally in the
range from about 0.1 .mu.M to about 1 .mu.M.
[0115] Furthermore, the present invention permits the skilled
artisan to design molecules, for example, selective protein
synthesis inhibitors that are tailored to be more potent with
respect to ribosomes of a target organism, for example, a pathogen
such a microbe, and less potent, i.e., less toxic, to ribosomes of
a non target organism, for example, host organism such as a human.
Also, the invention permits the skilled artisan to use the atomic
co-ordinates and structures of the large ribosomal subunit and its
complexes with protein synthesis inhibitors to design modifications
to starting compounds, such as an antibiotic, that will bind more
tightly to a target ribosome (e.g., the 50S ribosomal subunit of
bacteria) and less tightly to a non-targeted ribosome (e.g., human
60S ribosomal subunit or a human mitochondrial ribosome). For
example, it is contemplated that the present invention permits the
design of modified versions of cycloheximide, e.g., derivatives of
cycloheximide and other molecules, which bind selectively to
prokaryotic ribosomes, and which do not bind or do not bind as well
to the eukaryotic E site because of unfavorable interactions with
the L44E protein, or with the RNA residues of the eukaryotic E
site. As a second, alternative example, as part of a strategy to
design new antifungal agents, it is contemplated that modifications
of the dimethylcyclohexanone moiety of the cycloheximide core can
be designed which would enhance interactions with the fungal E-site
RNA and L44E protein.
[0116] The structure of a complex between the large ribosomal
subunit and the starting compound (e.g., cycloheximide) can also be
used to guide the modification of that compound to produce new
compounds that have other desirable properties for the applicable
industrial and other uses (e.g., as pharmaceuticals, herbicides or
insecticides), such as chemical stability, solubility or membrane
permeability.
[0117] The novel agents contemplated by the present invention can
be useful as herbicides, pesticides (e.g., insecticides,
nematocides, rodenticides, etc.), miticides, or antimicrobial
agents (e.g., antifungals, antibacterials, antiprotozoals, etc.) to
target specific organisms. For example, the novel agents can target
animal and plant parasitic nematodes, prokaryotic organisms
(disease causing microbes), and eukaryotic multicellular pests.
Specific examples of multicellular pests include, but are not
limited to, insects, fungi, bacteria, nematodes, mites and ticks,
protozoan pathogens, animal-parasitic liver flukes, and the
like.
[0118] Once a candidate molecule has been designed or selected by
the above methods, the affinity with which that molecule can bind
to the ribosome or ribosomal subunit can be tested and optimized by
computational evaluation and/or by testing biological activity
after synthesizing the compound. Candidate molecules can interact
with the ribosomes or ribosomal subunits in more than one
conformation, each of which has a similar overall binding energy.
In those cases, the deformation energy of binding can be considered
to be the difference between the energy of the free molecule and
the average energy of the conformations observed when the molecule
binds to the ribosomes or ribosomal subunits, more preferably to
the large ribosomal subunits, and even more preferably to the 50S
ribosomal subunits. A molecule designed or selected as binding to a
ribosome or ribosomal subunit can be further computationally
optimized so that in its bound state it preferably lacks repulsive
electrostatic interaction with the target region. Such
non-complementary (e.g., electrostatic) interactions include
repulsive charge-charge, dipole-dipole and charge-dipole
interactions.
[0119] Specific computer programs that can evaluate a compound
deformation energy and electrostatic interaction are available in
the art. Examples of suitable programs include: Gaussian 92,
revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa.); AMBER,
version 4.0 (P. A. Kollman, University of California at San
Francisco, Calif.); QUANTA/CHARMM (Molecular Simulations, Inc.,
Burlington, Mass.); OPLS-AA ("OPLS Force Fields." W. L. Jorgensen.
Encyclopedia of Computational Chemistry, Schleyer, Ed.; Wiley: New
York, 1998; Vol. 3, pp 1986-1989) and Insight II/Discover (Biosysm
Technologies Inc., San Diego, Calif.). These programs can be
implemented, for instance, using a Silicon Graphics workstation,
IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware
systems and software packages are known to those skilled in the
art.
[0120] Once a molecule of interest has been selected or designed,
as described above, substitutions can then be made in some of its
atoms or side groups in order to improve or modify its binding
properties. Generally, initial substitutions are conservative,
i.e., the replacement group will approximate the same size, shape,
hydrophobicity and charge as the original group. It should, of
course, be understood that components known in the art to alter
conformation should be avoided. Such substituted chemical compounds
can then be analyzed for efficiency of fit to the ribosome or
ribosomal subunit by the same computer methods described in detail,
above. In addition, once synthesized, the structure of the
candidate molecule when disposed within the 50S ribosomal subunit
can be determined by X-ray diffraction studies. Based on the
resulting structures, the skilled artisan can use this information
to modify and optimize the candidate molecule using the procedures
described herein.
[0121] 3. Synthesis of Candidate Molecules
[0122] A candidate molecule of the present invention can be, but is
not limited to, at least one selected from a lipid, nucleic acid,
peptide, small organic or inorganic molecule, chemical compound,
element, saccharide, isotope, carbohydrate, imaging agent,
lipoprotein, glycoprotein, enzyme, analytical probe, and an
antibody or fragment thereof, any combination of any of the
foregoing, and any chemical modification or variant of any of the
foregoing. In addition, a lead molecule can optionally comprise a
detectable label. Such labels include, but are not limited to,
enzymatic systems, radioisotopes, and spectroscopic labels, for
example, fluorescent labels, spin labels, chemiluminescent
compounds and bioluminescent compounds.
[0123] Methods useful for synthesizing lead molecules such as
lipids, nucleic acids, peptides, small organic or inorganic
molecules, chemical compounds, elements, saccharides, isotopes,
carbohydrates, imaging agents, lipoproteins, glycoproteins,
enzymes, analytical probes, antibodies, and antibody fragments are
well known in the art. Such methods include the traditional
approach of synthesizing one such candidate molecule at a time, as
well as the simultaneous synthesis of multiple candidate molecules
by combinational chemistries. Methods for the simultaneous
synthesis of multiple candidate molecules are particularly useful
in preparing combinatorial libraries, which can be used in
screening techniques known in the art.
[0124] 4. Characterization of Candidate Molecules
[0125] Candidate molecules designed, selected and/or optimized by
methods described above, once produced, can be characterized using
a variety of assays known to those skilled in the art to determine
whether the candidate molecules have biological activity. For
example, the candidate molecules can be characterized by
conventional assays, including but not limited to those assays
described below, to determine whether they have a predicted
activity, binding activity and/or binding specificity.
[0126] Furthermore, high-throughput screening can be used to speed
up analysis using such assays. As a result, it can be possible to
rapidly screen candidate molecules for activity, for example, as
anti-cancer, anti-bacterial, anti-fungal, anti-parasitic or
anti-viral agents. Also, it can be possible to assay how the
candidate molecules interact with a ribosome or ribosomal subunit
and/or are effective as modulators (for example, inhibitors) of
protein synthesis using techniques known in the art. General
methodologies for performing high-throughput screening are
described, for example, in Devlin (1998) High Throughput Screening,
Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays
can use one or more different assay techniques including, but not
limited to, those described below.
[0127] (1) Surface Binding Studies.
[0128] A variety of binding assays can be useful in screening new
molecules for their binding activity. One approach includes surface
plasmon resonance (SPR) which can be used to evaluate the binding
properties molecules of interest with respect to a ribosome,
ribosomal subunit or a fragment thereof.
[0129] SPR methodologies measure the interaction between two or
more macromolecules in real-time through the generation of a
quantum-mechanical surface plasmon. One device, (BIAcore
Biosensor.RTM. from Pharmacia Biosensor, Piscatawy, N.J.) provides
a focused beam of polychromatic light to the interface between a
gold film (provided as a disposable biosensor "chip") and a buffer
compartment that can be regulated by the user. A 100 nm thick
"hydrogel" composed of carboxylated dextran which provides a matrix
for the covalent immobilization of analytes of interest is attached
to the gold film. When the focused light interacts with the free
electron cloud of the gold film, plasmon resonance is enhanced. The
resulting reflected light is spectrally depleted in wavelengths
that optimally evolved the resonance. By separating the reflected
polychromatic light into its component wavelengths (by means of a
prism), and determining the frequencies which are depleted, the
BIAcore establishes an optical interface which accurately reports
the behavior of the generated surface plasmon resonance. When
designed as above, the plasmon resonance (and thus the depletion
spectrum) is sensitive to mass in the evanescent field (which
corresponds roughly to the thickness of the hydrogel). If one
component of an interacting pair is immobilized to the hydrogel,
and the interacting partner is provided through the buffer
compartment, the interaction between the two components can be
measured in real time based on the accumulation of mass in the
evanescent field and its corresponding effects of the plasmon
resonance as measured by the depletion spectrum. This system
permits rapid and sensitive real-time measurement of the molecular
interactions without the need to label either component.
[0130] (2) Fluorescence Polarization.
[0131] Fluorescence polarization (FP) is a measurement technique
that can readily be applied to protein-protein, protein-ligand, or
RNA-ligand interactions in order to derive IC.sub.50s and Kds of
the association reaction between two molecules. In this technique
one of the molecules of interest is conjugated with a fluorophore,
usually the smaller molecule in the system (in this case, the
molecules of interest). The sample mixture, containing the
molecule-probe conjugate and the ribosome, ribosomal subunit or
fragment thereof, is excited with vertically polarized light. Light
is absorbed by the probe fluorophores, and re-emitted a short time
later. The degree of polarization of the emitted light is measured.
Polarization of the emitted light is dependent on several factors,
but most importantly on viscosity of the solution and on the
apparent molecular weight of the fluorophore. With proper controls,
changes in the degree of polarization of the emitted light depends
only on changes in the apparent molecular weight of the
fluorophore, which in-turn depends on whether the probe-ligand
conjugate is free in solution, or is bound to a receptor. Binding
assays based on FP have a number of important advantages, including
the measurement of IC.sub.50s and Kds under true homogenous
equilibrium conditions, speed of analysis and amenity to
automation, and ability to screen in cloudy suspensions and colored
solutions.
[0132] (3) Cellular and Subcellular Assays.
[0133] It is contemplated that, in addition to characterization by
the foregoing biochemical assays, the molecules of interest can
also be characterized as a modulator (for example, an inhibitor of
protein synthesis) of the functional activity of the ribosome or
ribosomal subunit.
[0134] Furthermore, more specific protein synthesis inhibition
assays can be performed by administering the molecule to a whole
organism, tissue, organ, organelle, cell, a cellular or subcellular
extract, or a purified ribosome preparation and observing its
pharmacological and inhibitory properties by determining, for
example, its inhibition constant (IC.sub.50) for inhibiting protein
synthesis. Incorporation of .sup.3H leucine or .sup.35S methionine,
or similar experiments can be performed to investigate protein
synthesis activity. A change in the amount or the rate of protein
synthesis in the cell in the presence of a molecule of interest
indicates that the molecule is a modulator of protein synthesis. A
decrease in the rate or the amount of protein synthesis indicates
that the molecule is a inhibitor of protein synthesis.
[0135] Furthermore, the compounds can be assayed for
anti-proliferative or anti-infective properties on a cellular
level. For example, where the target organism is a micro-organism,
the activity of molecules of interest can be assayed by growing the
micro-organisms of interest in media either containing or lacking
the molecule. Growth inhibition can be indicative that the molecule
can be acting as a protein synthesis inhibitor. More specifically,
the activity of the molecules of interest against bacterial
pathogens can be demonstrated by the ability of the compound to
inhibit growth of defined strains of human pathogens. For this
purpose, a panel of bacterial strains can be assembled to include a
variety of target pathogenic species, some containing resistance
mechanisms that have been characterized. Use of such a panel of
organisms permits the determination of structure-activity
relationships not only in regards to potency and spectrum, but also
with a view to obviating resistance mechanisms. The assays can be
performed in microtiter trays according to conventional
methodologies as published by The National Committee for Clinical
Laboratory Standards (NCCLS) guidelines (NCCLS. M7-A5-Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; Approved Standard-Fifth Edition, NCCLS Document
M100-S12/M7 (ISBN 1-56238-394-9).
[0136] 5. Drug Formulation and Administration
[0137] The active molecules, once identified, can be useful in the
prevention or treatment of a variety of human or other animal
disorders, including for example, bacterial infection, fungal
infections, viral infections, parasitic diseases, and cancer. The
active molecules can be incorporated into any suitable carrier
prior to use. The dose of active molecule, mode of administration
and use of suitable carrier will depend upon the intended recipient
and target organism. The formulations, both for veterinary and for
human medical use, typically include such compounds in association
with a pharmaceutically acceptable carrier or excipient.
[0138] The carrier should be acceptable in the sense of being
compatible with the other ingredients of the formulations and not
deleterious to the recipient. Pharmaceutically acceptable carriers,
in this regard, are intended to include any and all solvents,
dispersion media, coatings, anti-bacterial and anti-fungal agents,
isotonic and absorption delaying agents, and the like, that are
compatible with pharmaceutical administration. The use of such
media and agents for pharmaceutically active substances is known in
the art. Supplementary active compounds (identified or designed
according to the invention and/or known in the art) also can be
incorporated into the formulations. The formulations can
conveniently be presented in dosage unit form and can be prepared
by any of the methods well known in the art of
pharmacy/microbiology. In general, some formulations are prepared
by bringing the active molecule into association with a liquid
carrier or a finely divided solid carrier or both, and then, if
necessary, shaping the product into the desired formulation.
[0139] A pharmaceutical composition of the invention should be
formulated to be compatible its intended route of administration.
Examples of routes of administration include oral or parenteral,
for example, intravenous, intradermal, inhalation, transdermal
(topical), transmucosal, and rectal administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide.
[0140] Useful solutions for oral or parenteral administration can
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., (1990). Formulations for
parenteral administration can also include glycocholate for buccal
administration, methoxysalicylate for rectal administration, or
citric acid for vaginal administration. The parenteral preparation
can be enclosed in ampuoles, disposable syringes or multiple dose
vials made of glass or plastic. Suppositories for rectal
administration also can be prepared by mixing the drug with a
non-irritating excipient such as cocoa butter, other glycerides, or
other compositions which are solid at room temperature and liquid
at body temperatures. Formulations also can include, for example,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, hydrogenated naphthalenes, and the like. Formulations for
direct administration can include glycerol and other compositions
of high viscosity. Other potentially useful parenteral carriers for
these drugs include ethylene-vinyl acetate copolymer particles,
osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation administration can contain as
excipients, for example, lactose, or can be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or oily solutions for administration
in the form of nasal drops, or as a gel to be applied
intranasally.
[0141] Formulations of the present invention suitable for oral
administration can be in the form of: discrete units such as
capsules, gelatin capsules, sachets, tablets, troches, or lozenges,
each containing a predetermined amount of the drug; a powder or
granular composition; a solution or a suspension in an aqueous
liquid or non-aqueous liquid; or an oil-in-water emulsion or a
water-in-oil emulsion. The drug can also be administered in the
form of a bolus, electuary or paste. A tablet can be made by
compressing or molding the drug optionally with one or more
accessory ingredients. Compressed tablets can be prepared by
compressing, in a suitable machine, the drug in a free-flowing form
such as a powder or granules, optionally mixed by a binder,
lubricant, inert diluent, surface active or dispersing agent.
Molded tablets can be made by molding, in a suitable machine, a
mixture of the powdered drug and suitable carrier moistened with an
inert liquid diluent.
[0142] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients. Oral
compositions prepared using a fluid carrier for use as a mouthwash
include the compound in the fluid carrier and are applied orally
and swished and expectorated or swallowed. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be
included as part of the composition. The tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose; a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring.
[0143] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). It should be stable under the
conditions of manufacture and storage and should be preserved
against the contaminating action of microorganisms such as bacteria
and fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0144] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation include vacuum
drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0145] Formulations suitable for intra-articular administration can
be in the form of a sterile aqueous preparation of the drug which
can be in microcrystalline form, for example, in the form of an
aqueous microcrystalline suspension. Liposomal formulations or
biodegradable polymer systems can also be used to present the drug
for both intra-articular and ophthalmic administration.
[0146] Formulations suitable for topical administration, including
eye treatment, include liquid or semi-liquid preparations such as
liniments, lotions, gels, applicants, oil-in-water or water-in-oil
emulsions such as creams, ointments or pastes; or solutions or
suspensions such as drops. Formulations for topical administration
to the skin surface can be prepared by dispersing the drug with a
dermatologically acceptable carrier such as a lotion, cream,
ointment or soap. Particularly useful are carriers capable of
forming a film or layer over the skin to localize application and
inhibit removal. For topical administration to internal tissue
surfaces, the agent can be dispersed in a liquid tissue adhesive or
other substance known to enhance adsorption to a tissue surface.
For example, hydroxypropylcellulose or fibrinogen/thrombin
solutions can be used to advantage. Alternatively, tissue-coating
solutions, such as pectin-containing formulations can be used.
[0147] For inhalation treatments, inhalation of powder
(self-propelling or spray formulations) dispensed with a spray can,
a nebulizer, or an atomizer can be used. Such formulations can be
in the form of a fine powder for pulmonary administration from a
powder inhalation device or self-propelling powder-dispensing
formulations. In the case of self-propelling solution and spray
formulations, the effect can be achieved either by choice of a
valve having the desired spray characteristics (i.e., being capable
of producing a spray having the desired particle size) or by
incorporating the active ingredient as a suspended powder in
controlled particle size. For administration by inhalation, the
compounds also can be delivered in the form of an aerosol spray
from pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0148] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants generally are known in the art,
and include, for example, for transmucosal administration,
detergents and bile salts. Transmucosal administration can be
accomplished through the use of nasal sprays or suppositories. For
transdermal administration, the active compounds typically are
formulated into ointments, salves, gels, or creams as generally
known in the art.
[0149] The active compounds can be prepared with carriers that will
protect the compound against rapid elimination from the body, such
as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. Liposomal suspensions can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0150] Where adhesion to a tissue surface is desired, the molecule
can include the drug dispersed in a fibrinogen-thrombin composition
or other bioadhesive. The molecule then can be painted, sprayed or
otherwise applied to the desired tissue surface. Alternatively, the
molecules can be formulated for parenteral or oral administration
to humans or other mammals, for example, in therapeutically
effective amounts, e.g., amounts which provide appropriate
concentrations of the drug to target tissue for a time sufficient
to induce the desired effect.
[0151] Active molecules identified or designed by the methods
described herein can be administered to individuals to treat
disorders (prophylactically or therapeutically). In conjunction
with such treatment, pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) can be considered.
Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician can consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a drug as well as tailoring the dosage and/or
therapeutic regimen of treatment with the drug.
[0152] In therapeutic use for treating, or combating, bacterial
infections in mammals, the molecules or pharmaceutical compositions
thereof will be administered orally, parenterally and/or topically
at a dosage to obtain and maintain a concentration, that is, an
amount, or blood-level or tissue level of active component in the
animal undergoing treatment which will be anti-microbially
effective. To be microbially effective, the molecule is present in
or on the recipient in an amount sufficient to elicit biological
activity, for example, anti-microbial activity, anti-fungal
activity, anti-viral activity, anti-parasitic activity,
anti-proliferative activity, anti-inflammatory activity, or
ameliorating a symptom of a gastrointestinal motility disorder.
Generally, an effective amount of dosage of active molecule will be
in the range of from about 0.1 to about 100, more preferably from
about 1.0 to about 50 mg/kg of body weight/day. The amount
administered will also likely depend on such variables as the type
and extent of disease or indication to be treated, the overall
health status of the particular patient, the relative biological
efficacy of the compound delivered, the formulation of the drug,
the presence and types of excipients in the formulation, and the
route of administration. Also, it is to be understood that the
initial dosage administered can be increased beyond the above upper
level in order to rapidly achieve the desired blood-level or tissue
level, or the initial dosage can be smaller than the optimum and
the daily dosage can be progressively increased during the course
of treatment depending on the particular situation. If desired, the
daily dose can also be divided into multiple doses for
administration, for example, two to four times per day.
[0153] In light of the foregoing general discussion, the specific
examples presented below are illustrative only and are not intended
to limit the scope of the invention. Other generic and specific
configurations will be apparent to those persons skilled in the
art.
III. EXAMPLES
Example 1
Three-Dimensional Structure of Cycloheximide Associated with the
Large Ribosomal Subunit
[0154] Ribosomes were purified and crystallized as described
previously (U.S. Patent Application Publication No. US 2002/0086308
A1). Crystals containing H. marismortui large ribosomal subunits
complexed with cycloheximide were obtained by soaking pre-formed,
large-subunit crystals in stabilizing buffers containing
cycloheximide. The cycloheximide was solubilized in
dimethylsulfoxide (DMSO), then added to the standard stabilization
buffer (Ban et al., (2000) Science, 289, 905-920 to a final
concentration of about 30.0 mM (and final DMSO concentration of
1%), and then incubated at 4.degree. C. for 24 hours prior to
cryo-vitrification of crystals in liquid propane. Initial X-ray
diffraction data were collected at beamline 8-BM of the Advanced
Photon Source, Argonne National Laboratory. Data were integrated
and scaled using the HKL2000 software package (Otwinowski (1997)
"Processing of X-ray Diffraction Data Collected In Oscillation
Mode," Methods in Enzymology 276(A):307-326).
[0155] Electron density corresponding to the antibiotic was first
seen in F.sub.o (antibiotic)-F.sub.e (native) difference Fourier
maps at 3.0 .ANG. resolution. The antibiotic model initially was
fit into F.sub.o-F.sub.c difference electron density maps, and then
the co-ordinates of the entire complex were refined. The structures
of these complexes were refined using CNX (Brunger et al. (1998)
Acta Cryst. D54: 905-921) for rigid body refinement, energy
minimization, and B-factor refinement of the entire native ribosome
structure including the antibiotic, and by a simulated annealing
refinement which allowed modifications of only a volume centered on
the bound cycloheximide. Nonisomorphous differences distant from
the antibiotic were ignored. The refinement process then was
repeated iteratively on the cycloheximide-containing model. The
atomic co-ordinates defining the structure of cycloheximide when
bound to the large ribosomal subunit are recorded on compact disk,
Disk No. 1 under file name cycloheximide.pdb.
INCORPORATION BY REFERENCE
[0156] The entire disclosure of each of the patent documents,
scientific articles, and atomic coordinates (including, without
limitation, those sets contained on Disk No. 1 as submitted in U.S.
Ser. No. 10/858,159) referred to herein is incorporated by
reference, herein for all purposes as though the content of each of
the patent documents, scientific articles, and atomic co-ordinates
were included herein.
[0157] Disk No. 1 was created on May 13, 2003 and is identified as
containing the following 2 files:
Disk No. 1:
TABLE-US-00003 [0158] File: Size (in bytes) FOLDERA: <DIR> --
<DIR> -- 1JJ2.pdb 8,270,586 FOLDERB: <DIR> --
<DIR> -- cycloheximide.pdb 7,588,893
EQUIVALENTS
[0159] The invention may be embodied in other specific forms
without departing form the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
* * * * *