U.S. patent application number 10/153130 was filed with the patent office on 2002-11-21 for anti-picornaviral ligands via a combinatorial computational and synthetic approach.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Cheh, James Li-wen, Hogle, James M., Isaacs, Lyle D., Joseph-McCarthy, Diane M., Karplus, Martin, Whitesides, George M..
Application Number | 20020172942 10/153130 |
Document ID | / |
Family ID | 22088054 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020172942 |
Kind Code |
A1 |
Joseph-McCarthy, Diane M. ;
et al. |
November 21, 2002 |
Anti-picornaviral ligands via a combinatorial computational and
synthetic approach
Abstract
The present invention provides structure-based combinatorial
libraries of compounds containing the functional group minima of
picornaviruses including poliovirus and rhinovirus. The libraries
can be used to screen for therapeutical antiviral compounds, e.g.,
anti-picornaviral capsid-binding compounds.
Inventors: |
Joseph-McCarthy, Diane M.;
(Belmont, MA) ; Isaacs, Lyle D.; (Cambridge,
MA) ; Whitesides, George M.; (Newton, MA) ;
Karplus, Martin; (Cambridge, MA) ; Hogle, James
M.; (Newton, MA) ; Cheh, James Li-wen;
(Somerville, MA) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
22088054 |
Appl. No.: |
10/153130 |
Filed: |
May 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10153130 |
May 21, 2002 |
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09210282 |
Dec 11, 1998 |
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6416762 |
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60069300 |
Dec 11, 1997 |
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Current U.S.
Class: |
435/5 ; 435/6.13;
435/7.1; 436/518; 544/265; 544/296; 568/58 |
Current CPC
Class: |
Y10T 436/141111
20150115; Y10T 436/147777 20150115; Y10T 436/14 20150115; A61K
47/6901 20170801 |
Class at
Publication: |
435/5 ; 435/6;
435/7.1; 436/518; 568/58; 544/265; 544/296 |
International
Class: |
C12Q 001/70; C12Q
001/68; G01N 033/53; G01N 033/543; C07D 43/14; C07D 473/02 |
Claims
What is claimed is:
1. A composition comprising a mixture of reaction products
resulting from a combinational synthesis of two aromatic monomers
and a spacer monomer, wherein the two aromatic monomers are
covalently linked through X to the spacer monomer, X is S or 0, the
aromatic monomer and the spacer monomer are functional group minima
of a picornavirus.
2. The composition according to claim 1 wherein the aromatic
monomer and the spacer monomer are functional group minima of
poliovirus.
3. The composition according to claim 1 wherein the aromatic
monomer and the spacer monomer are functional group minima of
rhinovirus.
4. The composition according to claim 1 wherein the two aromatic
monomers are selected from the group consisting of a single five or
six membered rings and fused double rings comprising a five
membered ring and a six membered ring or two six membered rings and
wherein one of the rings contains a hydroxyl group substituent or a
sulfhydryl group substituent.
5. The composition according to claim 1 wherein the spacer monomer
is a symmetric bis- (bromo methyl) arene or alkene.
6. The compositions according to claim 1 wherein the spacer monomer
is a bis (chloro methyl) arene or alkene.
7. The compositions according to claim 1 wherein the spacer monomer
is a bis (iodo methyl) arene or alkene.
8. The compositions according to claim 1 wherein the spacer monomer
is a bis (tosyl) arene or alkene.
9. The composition according to claim 1 wherein the aromatic
monomer contains a sulfyhydryl group substituent and the spacer
monomer is an .alpha., .omega. dibromo alkane.
10. The composition according to claim 1 wherein the aromatic
monomer contains a sulfyhydryl group substituent and the spacer
monomer is an .alpha., .omega. dichloro alkane.
11. The composition according to claim 1 wherein the aromatic
monomer contains a sulfyhydryl group substituent and the spacer
monomer is an .alpha., .omega.diido alkane.
12. The composition according to claim 4 wherein the aromatic
monomer containing the sulfyhydryl or hydroxyl substituent has one
or more additional substituents on the ring whereby the additional
substituents are less electophillic than the spacer monomers, e.g.,
these additional substituents may be hydroxyl, sulfhydryl, methoxy,
ester, nitro or halogens (such as fluro, chloro, bromo, iodo
groups).
13. The composition according to claim 4 wherein the structures
with the hydroxyl group may contain heteroatoms selected from the
group consisting of nitrogen, sulphur and oxygen.
14. The composition according to claim 12 wherein the ring
structures with the hydroxyl group may contain heteroatoms selected
from the group consisting of nitrogen, sulphur and oxygen.
15. The composition according to claim 4 wherein the ring
structures with the sulfhydryl group may contain heroatoms selected
from the group consisting of nitrogen and oxygen.
16. The composition according to claim 12 wherein the ring
structures with the hydroxyl group may contain heteroatoms selected
from the group consisting of nitrogen and oxygen.
17. The composition according to claim 4 wherein the spacer monomer
is linked to the aromatic ring structures by a sulfur or oxygen
atom.
18. A library comprising functional group minima of picornaviruses
covalently linked such that aromatic functional groups are linked
via linker functional groups and wherein the aromatic functional
groups are selected from the group consisting of a single five or
six membered rings and fused double rings comprising a five
membered ring and a six membered ring or two six membered rings and
wherein one of the rings contains a hydroxyl group substituent or a
sulfhydryl group substituent.
19. The library according to claim 18 wherein the spacer monomer is
a symmetric bis-(bromo methyl) arene or alkene.
20. The library according to claim 18 wherein the spacer monomer is
a bis (chloro methyl) arene or alkene.
21. The library according to claim 18 wherein the spacer monomer is
a bis (iodo methyl) arene or alkene.
22. The library according to claim 18 wherein the spacer monomer is
a bis (tosyl methyl) arene or alkene.
23. The library according to claim 18 wherein the aromatic monomer
contains a sulfhydryl group substituent and the spacer monomer is
an .alpha., .omega. dibromo alkane.
24. The library according to claim 18 wherein the aromatic monomer
contains a sulfhydryl group substituent and the spacer monomer is
an .alpha., .omega. dichloro alkane.
25. The library according to claim 18 wherein the aromatic monomer
contains a sulfhydryl group substituent and the spacer monomer is
an .alpha., .omega. diido alkane.
26. The library according to claim 18 wherein the aromatic monomer
contains a sulfhydryl or hydroxyl substituent has one or more
additional substituents on the ring whereby the additional
substituents are less electrophillic than the spacer monomers,
e.g., these additional substituents may be hydroxyl, sulfhydryl,
methoxy, ester, nitro or halogens (such as fluoro, chloro, bromo,
iodo groups).
27. The library according to claim 18 wherein the ring structures
with the hydroxyl group may contain heteroatoms selected from the
group consisting of nitrogen, sulphur and oxygen.
28. The library according to claim 26 wherein the ring structures
with the hydroxyl group may contain heteroatoms selected from the
group consisting of nitrogen, sulphur and oxygen.
29. The library according to claim 18 wherein the ring structures
with the sulfhydryl group may contain heteroatoms selected form the
group consisting of nitrogen and oxygen.
30. The library according to claim 26 wherein the ring structures
with the sulfhydryl group may contain heteroatoms selected form the
group consisting of nitrogen and oxygen.
31. The library according to claim 19 wherein the structure with
the hydroxyl group contains one or more substituents whereby the
substituents are non-electrophillic.
32. The library according to claim 18 wherein the spacer monomer is
linked to the aromatic monomer by a sulfur or oxygen atom.
33. The library according to claim 18 wherein the library contains
about 75, 75, or 275 members.
34. A method of making the library according to claim 4 comprising
the steps of: mixing one or more dissolved aromatic monomers to
form a mixture, incubating a solution of one or more dibromide
spacer monomers with the mixture to form an organic phase, washing
and concentrating the organic phase.
35. A method of making the library according to claim 34 comprising
the steps of: dispensing a stoichiometric amount of a dibromide
spacer monomer into each well of a microtiter plate or any
container having a microtiter plate like well arrangement,
dispensing a stoichiometric amount of a single pre-determined
aromatic monomer into each well of a vertical column, and
dispensing a stoichiometric amount of a single pre-determined
aromatic monomer into each well of a horizontal row 2.
36. A compound that binds to a Mahoney poliovirus capsid
represented by a structure selected from the group consisting of
those structures presented in FIG. 4a-h.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/069300, filed Dec. 11, 1997.
[0002] This work was partially supported by the U.S. government
under NIH grants U01 AI-32480, R37 AI-20566 and R01-GM30367. The
U.S. government retains certain rights in this invention.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention is broadly directed to structure-based
combinatorial libraries, especially a group of compounds containing
the functional group minima in picornaviruses including poliovirus
and rhinovirus. The libraries provided by the present invention
contain candidates of anti-picornaviral capsid-binding compounds
which can be therapeutically effective in antiviral treatments.
[0005] 2. Description of Related Art
[0006] Picornaviruses represent a very large virus family of small
ribonucleic acid-containing viruses responsible for many serious
human and animal diseases (Rueckert, R. R. Virology, 2nd ed.
(Fields, B. N. et al., eds.) Raven Press, Ltd., New York, p.
508-548 (1982)). Picornaviruses include four major groups:
enteroviruses, rhinoviruses, cardioviruses and apthoviruses.
Enteroviruses include polioviruses, Coxsackieviruses, echoviruses,
and four numbered enteroviruses.
[0007] Poliovirus is the etiologic agent of the disease
poliomyelitis in humans, and there are three known serotypes of the
virus. The oral poliovaccine, typically given to children, is a
mixture of the Sabin strain of the three serotypes of the virus.
Mahoney and Leon (the parent strains of Sabin 1 and 3,
respectively) are human neurovirulent strains of poliovirus. The
oral poliovirus vaccine is safe and effective, yet has two
limitations. First, the vaccine is unstable; current vaccines are
inactivated by relatively brief (less than 24 hours) exposure to
temperatures of 37.degree. C., necessitating transport in a frozen
state from the site of manufacture to the locale where they are
administered. Second, the vaccine occasionally reverts to virulence
in vaccine recipients; the reverted virulent virus may also be
passed to other individuals who come into contact with the
recipient in whom the vaccine has reverted.
[0008] The human rhinoviruses consist of at least 100 serotypes and
are the primary causative agents of the common cold. Because of the
large number of serotypes, development of a vaccine is problematic;
antiviral agents may therefore be the best approach to treatment.
The Coxsackieviruses (24 group A serotypes, 6 group B serotypes),
echoviruses (34 serotypes) and human enteroviruses (four
serotypes), are associated with a wide range of human diseases
including summer flus, diarrhea, meningitis, hepatitis, pneumonia,
myocarditis, pericarditis, and diabetes (Melnick, J. L. Virology,
2nd ed. (Fields, B. N. et al., eds.) Raven Press, Ltd., New York
p549-605). These infections occur sporadically in the general
population, but are becoming more common among children in day care
and their parents and siblings. Other important members of the
picornavirus family include human hepatitis A virus, Theiler's
murine encephalomyelitis virus, foot-and-mouth disease virus, and
mengovirus.
[0009] Several crystal structures of poliovirus and rhinovirus
capsids have been solved by X-ray diffraction. The X-ray structures
of poliovirus P1/Mahoney (Hogle, J. M., et al., Science 229:1358
(1985)); poliovirus P3/Sabin (Filman, D. J., et al., EMBO J. 8:1567
(1989)); rhinovirus 14 (Rossman, M. G., et al., Nature 317:145
(1985)); rhinovirus 1A (Smith, T. J., et al., Science 233:1286
(1986)); and rhinovirus 16 (Oliveira, M. A., et al., Structure
1(1):51-68 (1993)) are strikingly similar, although poliovirus and
the rhinoviruses are currently classified in different genuses.
Experimental results have revealed that there is a binding site in
the poliovirus structure which usually binds a lipid-like molecule
(Filman, D. J., et al., EMBO J. 8:1567 (1989)). When a drug is
bound in this site in poliovirus or rhinovirus, the virus is
stabilized, and in some cases, infection is prevented (McSharry, J.
J., et al., Virology 97:307 (1979); Smith, T. J., et al., Science
233:1286 (1986); reviewed in Zhang, A., et al., Virology, 3:453
(1992)). The functional group minima of picornavirus capsid
proteins have been determined via computational analysis of a
ligand binding site (D. Joseph-McCarthy et al., 1997, Proteins
29:32).
[0010] The existing drugs which are used against the viruses
described above are only moderately effective. Available drugs are
typically effective against only a limited subset of the rhinovirus
serotypes. In general, the available drugs have either failed to
demonstrate sufficient prophylactic effect or are converted in the
body into inactive metabolites. Furthermore, current drugs have all
been derived from the same parent compound that was found through
large-scale random screening of known chemicals for activity
against the virus, a very expensive and time-consuming process. A
need continues for additional drugs with better efficacy, and with
efficacy against pathogenic picornaviruses.
SUMMARY OF INVENTION
[0011] It is an object of the invention to provide a composition
which is a candidate anti-picornavirus capsid-binding compound.
[0012] It is another object of the invention to provide a library
of compositions useful for screening for anti-picornavirus
capsid-binding compounds.
[0013] It is yet another object of the invention to provide methods
of making the compositions and the libraries of the compositions
provided in the present invention.
[0014] These and other objects of the invention are provided by one
or more of the embodiments provided below.
[0015] In one embodiment of the invention there is provided a
composition which comprises two aromatic monomers and a spacer
monomer, wherein the two aromatic monomers are covalently linked
through X to the spacer monomer, where X is S or O, and the
aromatic monomer and the spacer monomer are functional group minima
of picornaviruses.
[0016] In another embodiment of the invention there is provided a
method of making the composition provided by the present invention
which comprises the steps of mixing equimolar amounts of one or
more aromatic monomers to form a mixture, incubating a solution of
one or more dibromide spacer monomers with the mixture to form an
organic phase, washing and concentrating the organic phase.
[0017] The present invention provides libraries of compounds which
are candidates for anti-picornaviral capsid-binding compounds. Such
compounds provide therapeutic benefits in anti-picornaviral
treatment. The present invention also provides methods for making
such libraries.
BRIEF DESCRIPTION OF THE DRAWING
[0018] FIG. 1 shows a schematic of library ligands of the present
invention.
[0019] FIG. 2 shows a schematic of the specific monomers that are
positioned in the ligands.
[0020] FIG. 3 shows a schematic of the combinatorial synthesis
scheme of the present invention.
[0021] FIG. 4 shows structures of compounds that have been screened
from a library of the present invention that bind to the Mahoney
poliovirus capsid.
[0022] FIG. 5 shows a schematic drawing of the virus binding
assay.
[0023] FIG. 6 shows a mass spectroscopy analysis of drugs released
from virus.
[0024] FIG. 7 shows the members of Library 6.1 and mass
spectroscopy of a virus binding assay of that library.
[0025] FIG. 8 shows the members of Library 6.2 and mass
spectroscopy of a virus binding assay of that library.
[0026] FIG. 9 shows a plot of the results of an
immunoprecipitation-based viral inhibition assay.
[0027] FIG. 10 shows data from a set of viral inhibition
assays.
[0028] FIG. 11 shows an Arrhenius plot for virus and drug
complexes.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention is broadly directed to structure-based
combinatorial libraries containing anti-picornaviral capsid-binding
compounds. Such compounds can be therapeutically effective in
anti-picornaviral treatments.
[0030] The libraries provided by the present invention are designed
based on computational methods. For picornaviruses, e.g.,
poliovirus and rhinovirus, functional group minima for regions of
the known drug binding pockets, in the capsids, can be calculated
and mapped according to computer programs readily available in the
art, e.g., the Multiple Copy Simultaneous Search (MCSS) program
(Miranker et al., Proteins, 11: 29, 1991; Evensen et al., MCSSv2,
Harvard University, Cambridge, Mass., 1997).
[0031] In a typical MCSS run, N copies of a given functional group
are randomly distributed in a specified site, where N is usually
between 1000 and 10,000. Functional groups are typically simple
small molecules. A large number of functional groups are available
in the current implementation of MCSS to picornaviruses, e.g.,
poliovirus and rhinovirus, and additional functional groups can
easily be included.
[0032] Functional group minima can be determined via any means
known in the art. Using MCSS, copies of functional groups can be
simultaneously and independently energy minimized in the field of a
fixed protein, with a computer program readily available in the
art, e.g., a modified version of the program CHARMM. By the
time-dependent Hartree approximation (Elber, R., et al., J.Am.
Chem. Soc. 112:9161 (1990)), each copy of functional groups will
feel the full force field of the protein but the copies do not
interact with each other. Specifically, the copies of the group are
simultaneously subjected to steps of minimization, e.g., 500 steps
of steepest decent minimization, followed by steps of Powell
minimization, e.g., 500 steps of Powell minimization and then 9
cycles of 1000 steps of Powell minimization each, for a total of
10,000 minimization steps. Thereafter minimization, e.g., about
every 1000 steps of minimization, duplicate functional group minima
are discarded. In addition, functional group minima are deleted
from the system after each cycle except for the first, if their
interaction with the protein energy is too high as determined by a
series of user specified energy cutoffs. After the final cycle, the
remaining minima may be sorted by interaction energy and their
coordinates and interaction energy may be recorded to a file. Since
the protein competes with solvent for binding functional groups,
functional group minima whose free energy of binding to the protein
is less than their free energy of salvation are preferred for the
structure-based combinatorial libraries provided in the present
invention.
[0033] Different approaches could be taken to construct chemically
sensible drug molecule candidates for the structure-based
combinatorial library of the present invention. One approach is to
connect the desired functional group minima via placing linker
carbon atoms between selected minima and optimizing the position of
the link atoms. Typically, each library compound is composed of
three connected monomers, e.g., two aromatic monomers and a spacer
monomer. The specific monomers that are selected for each of the
three monomer positions of the compounds are determined to a large
extent by calculations of computer programs readily available in
the art, e.g., MCSS.
[0034] Monomers may be aromatic alcohols or aromatic sulfhydryls.
The general structure of the monomers are shown in FIGS. 1-3. The
aromatic ring structure may be a five-membered ring, a six membered
ring or a five membered ring fused to a six membered ring or two
six membered rings fused.
[0035] The aromatic alcohols may be monocyclic or polycyclic with
one or more substituents in the ortho, meta or para positions on
the ring. The substituents must be non-electrophilic relative to
the spacer (e.g., BrCH.sub.2RCH.sub.2Br) monomers, and may be
hydroxyl, methoxy, ester, nitro, or thiol groups; halogens (such as
Cl, Br, F, I); or carbon atoms. If one ring structure is utilized
it is a five or six membered ring. If two ring structures are
utilized, one may be a six membered ring and the second may be a
five membered ring fused to the six membered ring. Alternatively,
two six membered rings may be fused. The aromatic alcohol may also
have one or more heteroatoms (nitrogen, sulfur or oxygen)
substituted for carbon atoms within the ring(s). Monomers with
saturated ring structures, e.g., cyclohexyl or two fused cyclohexyl
rings, are also possible. The hydroxyl group substituent may be
attached to any of the available ring atoms.
[0036] In another embodiment of the present invention, aromatic
sulfhydryls may be utilized as the monomers in the library design.
The aromatic sulfhydryls may be heterocyclic and contain one or
more heteroatoms in a single ring structure. If one ring structure
is utilized it is a five or six membered ring. If two ring
structures are utilized, one may be a six membered ring and the
second a five membered ring, or alternatively, two six membered
rings may be fused together. Nitrogen and oxygen may be substituted
for carbon atoms. Further, substituents may be present on the
sulfhydryl aromatic ring structure as described above for the
aromatic alcohol monomers.
[0037] The monomers are covalently linked by spacer compounds as
shown in FIGS. 1-3. The monomers and spacer compound are linked
through an oxygen or sulphur atom.
[0038] The spacer compound is a symmetric bis-(bromo methyl) arene
or alkane. For the oxygen linked library there must be double bonds
one carbon removed from the bromide substituents. Since thiols are
more nucleophilic than alcohols, double bonds are not required in
the linkers for the thiol library. The bromides are expected to be
separated by a carbon chain length of 2 to 5 atoms. Further, there
may be other small groups attached to the aryl or allyl groups.
Such groups include hydroxyl, methoxy, methyl, ester, nitro, or
halogens. In place of dibromides, dichchlorides, -iodides, or
-tosylates could also be used as spacer monomers.
[0039] The synthesis of the library compounds can be carried out by
any means known in the art. For example, solution phase chemistry.
The combinatorial synthesis scheme is shown in FIG. 3. Usually,
equimolar amounts of one or more desired aromatic monomers will be
dissolved to form a solution mixture. An equimolar solution of one
or more desired spacer monomers, i.e., dibromide spacers will be
added to the solution mixture of the aromatic monomers in the
presence of K.sub.2CO.sub.3. The resulting mixture may be incubated
for a period of time, e.g., 24 hours. The precipitate formed during
the reaction, e.g., KBr can be filtered out and the resulting
solution can be concentrated to dryness at reduced pressure. The
residue may be partitioned, e.g., between EtOAc and aqueous
NaHCO.sub.3 (saturated). The organic phase may be further washed,
dried, and concentrated at high vacuum. Any unreacted starting
materials, e.g., dibromide could be removed from the mixture by
aqueous acid extraction to pull the basic library members into the
acid layer or by adding beads with a thiol, e.g., dithiothreitol
attached, or beads with a bromide or other halide attached.
[0040] The structure-based combinatorial libraries provided by the
present invention could also be synthesized using a split synthesis
approach so that up to three related compounds are separately
compartmentalized, i.e., library as array. The library as array may
be synthesized in microtiter plates or any other container having a
microtiter plate like arrangement. Usually, a stoichiometric amount
of K.sub.2CO.sub.3, two identical or different monomers, and a
dibromide spacer monomer is dispensed into each well of a plate.
The dispensing of monomers and spacers into each well of a plate
may be pre-coded by any means known in the art so that monomer and
spacer compositions of a compound in each well are known. For
example, a stoichiometric amount of a single pre-determined monomer
may be dispensed into each well of a single horizontal row in a
plate; while a stoichiometric amount of a single pre-determined
monomer is dispensed into each well of a single vertical column in
a plate; and a stoichiomatric amount of a single pre-determined
spacer is dispensed into each well of a plate As a result, each
well will contain up to three related compounds with the monomers
connected in three different ways, e.g., M1-Spacer-M2,
M1-Spacer-M1,and M2-Spacer-M2.
[0041] The viral binding ability of the compounds provided by the
present invention can be assessed by any means known in the art,
e.g., cell-based assays, immunoprecipitation assays, fluorescence
assays, and crystallographic assays. For example, the minimum
inhibitory concentration (MIC) of the compounds can be determined
via cell-based assays. An MIC value is usually measured by a
standard method, as described in Andries et al., (J. Virol,
64:1117-1123, 1990). Briefly, serial dilutions of the compound of
the present invention are added to solutions containing
approximately 100 tissue culture infectious doses of viruses, and
the resulting mixtures are added to subconfluent layers of HeLa
cells in a microtiter plate. The MIC is taken as the lowest
concentration of a compound that protects 50% of cells from cell
death or cytopathic effect. The lower the MIC value of a compound,
the higher efficacy of the compound and presumably the viral
binding ability of the compound.
[0042] The viral-binding compounds in the libraries of the current
invention can be used to stabilize capsids of picornaviruses and
other related viruses by binding to the virus. The compounds can
thus be used to stabilize unstable forms of virus for experimental
studies. The compounds can also be used to increase the stability,
particularly the thermal stability, of existing poliovirus vaccines
or vaccines for related viruses. In addition, the compounds can be
used to prevent viral changes necessary for cell entry. The
compounds can also be used to terminate replication of live vaccine
virus after sufficient time has elapsed to induce an immune
response in an individual, minimizing the risk of vaccines shedding
reverted virus which are neurovirulent. The compounds can
additionally be used for prophylactic treatment of non-vaccinated
family members of vaccines. The compounds can further be used for
prophylaxis and therapeutic treatment of infection with
rhinoviruses, enteroviruses, Coxsackieviruses, echoviruses, and
other picornaviruses with accessible binding sites.
[0043] The following examples are provided for exemplification
purposes only and are not intended to limit the scope of the
invention which has been described in broad terms above.
Example 1
[0044] This example demonstrates the synthesis of Library 1 as
shown in FIG. 1.
[0045] A mixture of a group of five desired monomers (FIG. 2a) at
0.6 mmol each was dissolved in 20 ml DMF under N.sub.2. Solid
K.sub.2CO.sub.3 of 0.829 g (6 mmol) was added to the solution which
was stirred at RT for 5 min. To the resulting mixture an equimolar
solution of a group of five dibromide spacer monomers (FIG. 2b) at
0.3 mmol in 15 ml DMF was added. The reaction was continued at room
temperature for 24 hours. The precipitated KBr was filtered out.
The resulting solution was concentrated to dryness at reduced
pressure. The residue was partitioned between EtOAc and aqueous
NaHCO.sub.3 (saturated). The organic phase of the solution was
further washed with H.sub.2O and aqueous NaCl (saturated.), dried
over MgSO.sub.4, and concentrated. The resulting amber colored
material was dried at high vacuum. Any unreacted dibromide could be
removed from the mixture by aqueous acid extraction (thereby
pulling the basic library members into the acid layer) or by adding
beads with a thiol (e.g., dithiothreitol) attached. Assuming that
all of the reactions occurred as planned, the library is expected
to consist of 75 1 [ Formula 1 : m i = 1 n i
[0046] where m is the number of spacer monomers (here 5) and n is
the number of thiol monomers (here 5)] compounds including all
possible unique combinations of the allowed monomers.
Example 2
[0047] This example demonstrates the synthesis of Library 2 as
shown in FIG. 1.
[0048] A mixture of five desired monomers (FIG. 2c) at 0.6 mmol
each and five desired dibromide spacers (FIG. 2b) at 0.3 mmol each
were dissolved in 4 ml DMF under N.sub.2. Solid K.sub.2CO.sub.3 of
0.415 g (3 mmol) was added to the solution which was subsequently
stirred at 57.degree. C. overnight. The reaction mixture was then
diluted with water and extracted with Et.sub.2O and EtOAc.
Subsequently, the organic phase of the reaction mixture was washed
with 2M NaOH, dried over K.sub.2CO.sub.3, and concentrated. The
resulting amber colored material was then dried at high vacuum.
Assuming that all of the reactions occurred as planned, the library
is expected to consist of 75 [Formula 1 above, where m is the
number of spacer monomers (here 5)] and n is the number of hydroxyl
monomers (here 5))compounds including all possible unique
combinations of the allowed monomers.
Example 3
[0049] This example demonstrates the synthesis of Library 3 as
shown in FIG. 1.
[0050] A mixture of five desired thiol monomers (FIG. 2a) at 0.6
mmol each, five desired phenol monomers (FIG. 2c) at 0.6 mmol each
as well as the five desired dibromide spacers (FIG. 2b) at 0.3 mmol
each were dissolved in 4 ml DMSO under N2. Solid K.sub.2CO.sub.3 of
0.829 g (6 mmol) was added to the solution which was then stirred
at 57.degree. C. overnight. The reaction mixture was subsequently
diluted with water, extracted with Et.sub.2O and EtOAc. The organic
phase of the reaction mixture was washed with 2M NaOH, dried over
K.sub.2CO.sub.3 and concentrated. The resulting amber colored
material was then dried at high vacuum. Assuming that all of the
reactions occurred as planned, the library is expected to consist
of 275 [Formula 1 (above), where m is the number of spacer monomers
(here 5) and n is the number of thiol monomers plus the number of
hydroxyl monomers (here 5+5=10)] compounds including all possible
combinations of the allowed monomers.
Example 4
[0051] This example demonstrates the synthesis of library as array
in microtiter plates.
[0052] The procedure to synthesize library as array in a 8.times.12
microtiter plate is as follows: 1) to each well of the plate,
adding a stoichiometric amount of K.sub.2CO.sub.3, it may be
necessary to make an aqueous solution to make the dispensing
easier, 2) to each well of a horizontal row, adding a
stoichiometric amount of a single pre-determined monomer, changing
the identity of the monomer for each horizontal row, 3) to each
well of a vertical column, adding a stoichiometric amount of a
single pre-determined monomer, changing the identity of the monomer
for each vertical column, 4) to each of the 96 wells, add a
stoichiometric amount of a single pre-determined spacer dibromide
in DMF (different dibromides would be used on separate 96 well
plates).
Example 5
[0053] This example confirms the activity of the combinatorial
libraries and demonstrates the screening procedures.
[0054] The library of 75 compounds described in Example 1 was
screened for potential binders to the Mahoney poliovirus capsid.
FIG. 2 shows the eight compounds found in the assay to screen for
viral binding compounds. The cell free assay is described as
follows.
[0055] 1) Formation of Virus-Compound Complexes.
[0056] The initial phase of the assay involves the incubation of
virus with libraries of compounds to allow for the specific
interaction between the compounds and virus. Poliovirus
(P1/Mahoney, or P1/M) is grown in HeLa cells and purified by
differential centrifugation and CsCl density gradient fractionation
according to standard methods (Rueckert, R. R. and Pallansch, M.,
Methods in Enzymol. 78:315-325 (1981)). The virus is stored frozen
in phosphate buffered saline (PBS) at -80.degree. C. until use.
Stock solutions of the virus are in PBS, while mixtures of
potential ligands are dissolved in DMSO. These stock solutions are
diluted such that all incubations are carried out at a final DMSO
concentration of 5% in PBS. Final volumes ranged from 0.5 to 1.0
mL. Once mixed, the incubations were left at room temperature for
one to four hours or kept at 4.degree. C. overnight (See FIG. 5 for
schematic).
[0057] The sensitivity of the assay is largely dictated by two
factors: (1) the amount of virus used, which determines the number
of available virus binding sites, and (2) the current detection
limit of mass spectroscopy (MS). For our electro-spray mass
spectrometer, this limit is 50 picomole/25 .mu.L or 2 .mu.M. In the
case of MALDI-TOF MS, the mass spectrometer at our disposal has a
limit of approximately 1 picomole. Using our MALDI-TOF MS,
therefore, to select ten compounds from a library of indefinite
size requires 10 picomoles of viral sites (see below for recovery
rates). A typical experiment requires between 0.1 and 0.8 mg of
virus in 0.5 mL incubation mixture.
[0058] Detection of virus-compound complex formation largely
depends upon two factors. First, the concentration of the compound
in the incubation must be above a critical threshold. Existing
anti-picornaviral compounds work in the micromolar to nanomolar
range. The estimated concentration of compound in the typical stock
solutions of existing libraries is 6.times.10.sup.-3M. Screens have
been carried out with the total concentration of compound in the
incubation ranging from 10 to 350 .mu.M. Complex formation can be
driven by increasing the number of drug molecules per viral binding
sites. In so doing, a competition results between compounds in the
library for the available binding sites. Those compounds which have
the highest affinity for the pocket, i.e., viral binding site will
easily compete out the weaker binders and the excess compound
ensures that the equilibrium of binding is shifted towards
complex.
[0059] 2) Purification of Virus-Compound Complexes.
[0060] Virus-drug mixtures are loaded onto a size exclusion column
(1.4 cm diameter.times.8.4 cm height) having a Sephacryl S-200 or
S-300 matrix (Pharmacia) to separate virus-drug complexes from
unbound drug. The running buffer is 5% DMSO in PBS. One mL
fractions are collected at 0.5 mL/min. Identification of fractions
containing the void volume, and therefore possibly containing
viruses bound with compound, is accomplished with a Pharmacia
Uvicord SII UV monitor which measures the OD280 of the material as
it is pumped out of the column (See FIG. 5 for schematic).
[0061] 3) Extraction and Concentration of Virus-Bound Drug.
[0062] Fractions containing virus bound with compounds are mixed
with a double volume of ethyl acetate, vortexed for 30 seconds,
then centrifuged for 10 minutes at 12,000 X g. At this point the
viruses will be completely denatured. Previously virus-bound drug
is released, and will partition into the ethyl acetate phase. The
upper organic phase is separated from the aqueous phases. The
organic phase containing the compounds is dried in a centrivap (See
FIG. 5 for schematic). 4) Preparation of Sample for MS.
[0063] A small volume (50 .mu.L) of an organic solvent is added to
the tube containing the dried sample. Which solvent should be used
depends upon the MS technique used for the analysis. For MALDI-TOF
MS, the solvent is acetonitrile or THF. In the case of
electro-spray MS, it is ethanol. After the addition of solvent, the
tube containing the sample is vortexed. At this point, the
concentration of each virus-bound compound is at least 2 .mu.M,
high enough for detection by electro-spray MS or MALDI-TOF MS. The
decision between electro-spray or MALDI-TOF depends upon the
material in the sample.
[0064] 5) Detection of Virus Bound Compounds.
[0065] A) Optimization of Assay Conditions with Radiolabeled
Compounds.
[0066] The conditions of this assay were optimized using the
radiolabeled compound R77975 (Janssen Pharmaceuticals). The
radiolabel enabled us to track the amount of drug recovered at each
step of the assay. Briefly, in a siliconized tube 240 mL of a
preparation containing 2.32 mg/mL of viral binding sites, i.e.,
P1/M in PBS was mixed with 15.8 mL of 2.5.times.10.sup.-4M
.sup.3H-R77975 in DMSO. The incubation volume was brought up to 500
mL and the DMSO content was 5% in PBS. The ratio of drug to viral
binding sites or pockets was 1:1. This mixture was left at room
temperature for 2 hours, then loaded onto the size exclusion
column. Fractions 6 through 11 contained the virus as determined by
UV trace.
[0067] To determine the yield of .sup.3H-R77975 in each fraction,
100 mL of each fraction was transferred to a 7 mL scinitillation
vial containing Ecoscint A (National Diagnostics) and counted in a
Beckman 500OTD scintillation counter. 33% of the input labeled
compound was found to be in the void volume and was bound to the
virus. Nearly 100% of this material was recovered after the ethyl
acetate separation. Subsequent concentration of this sample in the
centrivap yielded 77% of the extracted compound. The overall
recovery of drug was 26%.
[0068] To show that the binding of R77975 is specific, a
competition experiment was done with R78206, a tighter binding
compound, and the results showed that the assay is capable of
selecting the best binder out of a mixture of compounds. 57 .mu.g
of P1/Mahoney poliovirus was incubated in a PBS 5% DMSO solution
containing 4.times.10.sup.-9 moles of .sup.3 H-R77975 and R78206
respectively, corresponding to a drug to pocket ratio of 10:1 for
each compound. The concentration of drug used in the incubation was
8 .mu.M. Under these conditions only R78206 is expected to be bound
to virus since its minimum inhibitory concentration (MIC) is 8 nM,
compared to 3.mu.M for R77975. After an incubation time of 2 hours
at room temperature, the mixture was run through the column. By
counting the amount of radioactivity in the void volume, it was
possible to determine whether any of the R77975 bound to the virus.
As expected, there was no radioactivity associated with virus in
the void volume.
[0069] B) Assay Detects Binding of Drug with nM MIC.
[0070] R78206 (Janssen Pharmaceuticals), a more potent analog of
R77975, was incubated with viral binding sites, i.e., P1/M.
Previous work had shown that this compound bound to the virus
inside the hydrophobic pocket of VP 1 and prevented viral
replication in vitro. In this incubation, the moles of R78206
equaled the moles of pocket sites. The concentration of R78206 in
the incubation was 8 .mu.M. R78206 has a molecular weight of 383
and gave an [M+H]+peak of 384 in the MALDI-TOF mass spectrum from
this experiment. As an additional control, this experiment was
repeated with no virus present to ensure that the signal was not
simply due to some small amount of residual unbound drug, and as
expected, no peak was observed at 384.
[0071] C) Assay Detects Binders in a Mixture of Compounds with MIC
Values Ranging From nM to .mu.M.
[0072] In this experiment, three Janssen analogs, R77975, R78206,
and R80633, were incubated with virus. All compounds were present
in equimolar amounts and there were exactly enough sites to bind
all of each compound. To be more precise, there were
6.times.10.sup.-9 moles of binding sites and 2.times..sup.10.sup.-9
moles of each compound. In a 500 .mu.L volume, the drug
concentration was 4 .mu.M. After an overnight incubation at
4.degree. C., the incubation was assayed and the resulting
MALDI-TOF mass spectrum contained only three peaks corresponding to
the three compounds (FIG. 6).
[0073] D) Ability of Assay to Discriminate Between Binders and
Non-binders
[0074] To ensure that the binding of drug to virus was specific, we
constructed a test library containing two drugs with proven
anti-viral activity, R78206 and R80633, alongside a cocktail of
structurally related compounds with no expected ability to bind
virus (as shown in earlier crystallographic experiments). In this
incubation, each compound was present in an equimolar amount and
there were exactly enough pockets to bind all of each compound. The
MALDI-TOF spectrum from this experiment indicated the expected
peaks for R78206 and R80633. Of the other five compounds, a peak
was detected for one, indicating that it may in fact be a weak
binder. All other parent molecular weight ion peaks were absent. E)
Library A +virus.
[0075] A library of 75 potential ligands was synthesized using
combinatorial chemistry as described above. The design of this
library was based upon extensive computer modeling on poliovirus
and rhinovirus (Joseph-McCarthy, D. et al., Proteins 29:32-58
(1997)). An incubation was set up such that the concentration of
the library was 15-fold greater than the concentration of virus
binding sites. Specifically the concentration of library A was
3.24.times.10.sup.-4 M in the incubation, while the concentration
of virus sites was 2.16.times.10.sup.-5 M. Given the amount of
virus relative to compounds used in this assay, there should be
enough sites to bind up to approximately 5 compounds.
[0076] After subjecting this incubation to the binding assay, the
concentrated sample was analyzed by both electrospray and MALDI-TOF
MS. In the MALDI-TOF spectrum, there were in fact 5 peaks and 1
weak peak that could correspond to compounds in the library. The
electrospray spectrum showed five relatively strong peaks which
corresponded to compounds in the library as well, three of which
were also present in the MALDI-TOF spectrum. There were 4
additional weak peaks in the electrospray mass spectrum that could
correspond to compounds in the library, one of which also appeared
as a weak signal in the MALDI-TOF spectrum. In total, 7 reasonable
peaks from both sets of spectra that could correspond to library
compounds were identified. These individual compounds will now be
synthesized and confirmed for binding by using more stringent
assays (e.g., immunoprecipitation, cell-based, fluorescence, and
crystallography).
[0077] In order to confirm the results from the Library A screen, a
six compound library that is a subset of Library A was
combinatorially synthesized. This six compound library contains two
of the potential Library A binders from the previous screen. One
milligram of virus was incubated with a PBS/5% DMSO solution
containing 5.7.times.10.sup.-4M of the six compound library
(roughly 10 .mu.M per compound), corresponding to a library to
pocket ratio of 4:1. After an overnight incubation at 4.degree. C.,
the incubation was run through the size exclusion column and
fractions 7 to 13 were extracted, concentrated, and analyzed by
mass spectroscopy. The library is expected to contain compounds
with molecular weights 280, 292, 304, 367, 379, and 454. The
spectrum identified four potential binders.
[0078] F) A Hybrid Split-Pool Synthesis Approach.
[0079] An alternative approach to screening compounds in
combinatorial libraries involves the generation of multiple
libraries, each containing approximately 5 to 10 compounds. This
approach avoids difficulties in controlling the relative
representation of individual members of the larger (approximately
100 compound) libraries and the relatively low signal to noise
ratio in mass spectroscopic analyses when larger libraries are
used.
[0080] To test this approach, two small libraries containing six
compounds each, termed Libraries 6.1 and 6.2, respectively, were
synthesized (FIGS. 7 and 8) and tested for viral binding. The mass
spectroscopic assay provided convincing evidence that two compounds
from Library 6.1 and three compounds from Library 6.2 bind to the
virus (FIGS. 7 and 8). In addition to increased control over the
synthesis and improved signal to noise in the assay, the hybrid
split-pool approach has the advantage that each of the sublibraries
may be designed to ask a specific question about modification of a
portion of the candidate ligand. Indeed, Library 6.1 was originally
designed to explore the consistent hits from library A, and Library
6.2 was designed to address the role of large aromatic versus small
aliphatic linkers in the central region of the molecule.
[0081] Relatively large numbers of such small libraries may be
synthesized simultaneously, and the results analyzed by the sparse
matrix and limited factorial approaches that have become popular in
other complex multidimensional searches, including crystallization
(Schumacher, T. N. et al., 1996, Science 271: 1854; Shuker, S. B.,
et al., 1996, Science 274: 1531; Smith, T. J. et al., Science 233:
1286). The results of the first round could then be used to design
the next generation of multiple small libraries that either probe
promising areas of ligand space on progressively finer matrices, or
address problems (e.g. solubility or toxicity) which have been
identified in the previous round.
[0082] G) A Functional Assay for Screening Libraries.
[0083] Although the mass spectrometry assay has proved very useful
in identifying ligands that bind virus it does not address the
question of whether the observed binding is functional. Therefore,
a secondary functional assay was developed. A test for the ability
of compounds to inhibit viral infectivity, for example a tissue
culture assay such as that described by Grant, R. A. et al., 1994,
Curr. Biol. 4: 784 (the method of which is herein incorporated by
reference) may be used. However, this assay is delicate and
somewhat cumbersome and requires rigorous purification of ligands
to avoid toxicity of reactants and by-products. An indirect assay
that measures the ability of ligands to interfere with the
conformational conversion of the virus from its native, 160S state
to the 135S infective form has therefore been developed. The assay
is based on the observation that virus can be efficiently converted
to the 135S form in the absence of receptor by heating in hypotonic
buffers in the presence of low levels of Ca.sup.+2.
[0084] The assay involves immunoprecipitation using antibodies
directed against a peptide corresponding to residues 21-40 of VP1.
This region is exposed during the native-135S transition, such that
the antibody can only recognize the 135S, and not the native form
of the virus. Preliminary studies showed that the rate of
conversion of native (unliganded) virus varied steeply as a
function of temperature (FIG. 9) and that the rate is significantly
reduced by binding drugs with known antiviral activity. The assay
measures the rate of conversion at 43.degree. C. where the rates
for native virus and virus-R78206 complexes are both within the
practical limits of the assay. Briefly, radiolabelled virus is
incubated with a compound or library of compounds and then diluted
into prewarmed buffer at 43.degree. C. At several time points,
aliquots are removed and quenched in chilled buffer. Antisera
specific for the amino terminus of VP1 are then added and incubated
for 30 min at room temperature. Complexes of antibody with altered
virus are then precipitated with protein A beads. The precipitate
and supernatant are counted and the first order rate of conversion
is calculated from the slope of the best fit line in a plot of log
((total counts-counts precipitated)/total counts)vs time.
[0085] The immunoprecipitation assay has been used to characterize
the effect of the 6.1 and 6.2 libraries, as well as three
individual compounds from the 6.1 library, on the rate of the
native-135 S conversion. Two of the purified compounds (304 and
367) had been identified as virus binders in the mass spectroscopy
assay. The third (454) was not represented in the library. When
synthesized separately the 454 compound was shown to be highly
insoluble in most solvents tested, which probably accounts for its
failure to be produced in the original library. It was soluble in
0.1% methyl-pyrillodone. Both of the libraries (data for 6.1 are
shown) and the 304 and 367 compounds produced modest but highly
significant reductions in the rate of conversion at 43.degree. C.
(see Table 1 and FIG. 10). The individual compounds 304 and 367
showed more dramatic rate reductions. The rate decrease for the 454
compound is even more impressive (even when the protective effect
of the solvent is discounted), and the concentration quoted (50 uM)
is likely to be a gross over estimate of the true concentration of
the drug because of its very limited solubility.
[0086] H) Crystallographic Assay Confirms Binding in Normal Drug
Binding Pocket.
[0087] Crystallographic studies of complexes of the candidate
ligands with P1/M virus have been performed. To date nearly
complete data sets have been collected from crystals soaked in
candidate ligands 367 and 304. Data from the 367 complex have been
processed, and the structure solved by molecular replacement. The
resulting electron density maps clearly show that the ligand is
present in the pocket at high occupancy. This confirms that the
screening method of the invention is effective in the
identification of compounds which bind in the ligand binding pocket
of the virus.
[0088] I) Stabilization of Virus is Entropically Determined.
[0089] The very steep dependence of the first order rate of the
native to 135S transition on temperature implies that the
activation enthalpy is very high. Indeed, Arrhenius analysis of the
dependence of ln(k) vs 1/RT yields an activation enthalpy of
approximately 120 kcal/mole (FIG. 11). Previous models for the
mechanism of drug action had assumed that the drugs would work by
increasing the "stiffness" of the VP1, thereby increasing the
activation enthalpy and decreasing the rate of conversion. However,
when similar data are plotted for a variety of virus-complexes, the
data fall on parallel lines (FIG. 11), indicating that the enthalpy
of activation is identical (or nearly identical) to that of free
virus. This means that the predominant effect of drug binding on
rate of conversion is entropically derived. Phelps and Post, 1995,
J. Mol. Biol. 254: 544, reached a similar conclusion from molecular
dynamics simulations of free rhinovirus and rhinovirus drug
complexes.
[0090] J) Infectivity Inhibition Assay.
[0091] The definitive in vitro assay for antiviral activity is an
assay that measures a compound's ability to inhibit a virus'
ability to lyse cells in culture. A number of variations of this
assay have been developed and published. A version of the assay
used by Janssen was adapted for use in these screening approaches.
Briefly, serial five-fold dilutions of compounds are added directly
to a 96 well microtiter plate. Approximately 100 TCID50 of virus is
then added to each well and incubated at 37.degree. C. After two
hours 10.sup.4 HeLa cells are added to each well and the plate is
incubated for 3 days at 37.degree. C., then stained with crystal
violet. The minimum inhibitory concentration is defined as the
concentration of drug required to reduce the cytopathic effect by
50%.
[0092] The principles, preferred embodiments and modes of operation
of the present invention have been described in the foregoing
specification. The invention which is intended to be protected
herein, however, is not to be construed as limited to the
particular forms disclosed, since they are to be regarded as
illustrative rather than restrictive. Variations and changes may be
made by those skilled in the art without departing from the spirit
of the invention.
1TABLE 1 Rate Constants at 43.degree. C. Compound k(10.sup.-5
s.sup.-1) 0.1% 640 .+-. 23 40 .mu.M 77975 195 .+-. 3 12 .mu.M 6 cpd
550 .+-. 49 40 .mu.M 304 470 10 .mu.M 367 260 .+-. 27 1% methyl-
269.9 pyrillodone 50 .mu.M 454 62.8
* * * * *