U.S. patent application number 10/429915 was filed with the patent office on 2004-04-15 for method for identifying new anti-picornaviral compounds.
Invention is credited to Hogle, James M., Joseph-McCarthy, Diane M., Tsang, Simon K..
Application Number | 20040072149 10/429915 |
Document ID | / |
Family ID | 22088063 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072149 |
Kind Code |
A1 |
Tsang, Simon K. ; et
al. |
April 15, 2004 |
Method for identifying new anti-picornaviral compounds
Abstract
The present invention is broadly directed to methods of
screening viral-binding compounds. In particular, the present
invention provides cell-free assays to rapidly screen libraries of
compounds for viral-capsid binding activity. Such compounds are
useful for anti-viral treatments.
Inventors: |
Tsang, Simon K.; (Cambridge,
MA) ; Joseph-McCarthy, Diane M.; (Belmont, MA)
; Hogle, James M.; (Newton, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
22088063 |
Appl. No.: |
10/429915 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10429915 |
May 5, 2003 |
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09210118 |
Dec 11, 1998 |
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6558899 |
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60069302 |
Dec 11, 1997 |
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Current U.S.
Class: |
435/5 ;
435/7.1 |
Current CPC
Class: |
C12Q 1/70 20130101; G01N
2333/085 20130101; G01N 2333/105 20130101; G01N 2333/095 20130101;
G01N 33/56983 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
435/005 ;
435/007.1 |
International
Class: |
C12Q 001/70; G01N
033/53 |
Goverment Interests
[0002] This work was partially supported by the U.S. government
under NIH grants U01 AI-32480 and R37 AI-20566. The U.S. government
retains certain rights in this invention.
Claims
1. A method for screening viral-binding compounds comprising the
steps of: incubating in vitro one or more compounds with viral
sites, wherein binding of a compound to the viral site inhibits
viral infection, separating a viral site-bound compound from an
unbound compound, and detecting the viral site-bound compound.
2. The method of claim 1 wherein the viral site is contained within
a virus.
3. The method of claim 2 wherein the virus is a picornavirus.
4. The method of claim 3 wherein the picornavirus is selected from
the group consisting of poliovirus and rhinovirus, Coxsackie B
virus, Coxsackie A virus, echovirus, or human enterovirus.
5. The method of claim 1 wherein the viral site is contained within
a viral capsid.
6. The method of claim 5 wherein the viral site is a conserved
hydrophobic pocket at the core of VP1.
7. The method of claim 1 wherein the viral site-bound compound is
detected via mass spectroscopy.
8. The method of claim 1 wherein the number of compounds is less
than or equal to the number of viral sites.
9. The method of claim 8 wherein the number of compounds is in
excess of the number of viral sites.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/069302, filed Dec. 11, 1997.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention is broadly directed to methods of
screening viral-binding compounds. In particular, the present
invention provides cell-free assays to rapidly screen libraries of
compounds for viral-capsid binding activity. Such compounds are
useful for 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.
(Field, B. N. et al., eds.) Raven Press, Ltd., New York, p. 508-548
(1982)). Piconaviruses include four major genera: enteroviruses,
rhinoviruses, cardioviruses and aphthoviruses. The enterovirus
genus includes polioviruses and Coxsackieviruses, echoviruses, and
four numbered human enterovirus species.
[0007] Poliovirus is the etiologic agent of the disease
poliomyelitis in humans. The human rhinoviruses consist of at least
100 serotypes and are the primary causative agents of the common
cold. 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,
pneumonitis, 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. In general, the available
drugs have either failed to demonstrate sufficient prophylactic
effect or are converted in the body into inactive metabolites.
[0008] The 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. Such screening
procedure is a very expensive and time-consuming process. There is
a need in the art to develop quick and efficient assays to screen
for anti-viral compounds. In particular, there is a need in the art
to establish cell-free assays for screening viral-binding
compounds, e.g., picornaviral-binding compounds and libraries of
such compounds.
SUMMARY OF THE INVENTION
[0009] It is an object of the invention to provide a method for
screening viral-binding compounds.
[0010] This and other objects of the invention are provided by one
or more of the embodiments provided below.
[0011] In one embodiment of the invention there is provided a
method for screening viral-binding compounds which comprises the
steps of incubating in vitro one or more compounds with a viral
site, wherein binding of a compound to the viral site inhibits
viral infection, separating a viral site-bound compound from an
unbound compound, and detecting the viral site-bound compound. In a
preferred embodiment, the viral site is contained within a viral
capsid.
[0012] The present invention provides in vitro assays for rapid
screening of anti-viral compounds, especially viral-binding
compounds. Such compounds can be therapeutically effective in
anti-viral treatments.
BRIEF DESCRIPTION OF THE DRAWING
[0013] FIG. 1 shows a schematic drawing of the virus binding
assay.
[0014] FIG. 2 shows a mass spectroscopy analysis of drugs released
from virus.
[0015] FIG. 3 shows the members of Library 6.1 and mass
spectroscopy of a virus binding assay of that library.
[0016] FIG. 4 shows the members of Library 6.2 and mass
spectroscopy of a virus binding assay of that library.
[0017] FIG. 5 shows a plot of the results of an
immunoprecipitation-based viral inhibition assay.
[0018] FIG. 6 shows data from a set of viral inhibition assays.
[0019] FIG. 7 shows an Arrhenius plot for virus and drug
complexes.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides a high-throughput in vitro
assay that can rapidly screen libraries of compounds for specific
binding to a viral target. Libraries of hundreds or thousands of
compounds can be simultaneously screened by the cell-free assays
described in the present invention within a very short period of
time, e.g., a few hours. The viral-binding compounds detected by
the screening assays of the present invention are potential
therapeutic compounds for anti-viral treatments.
[0021] This method may be generally used to screen for binding to
any nonenveloped virus (including picornaviruses, calciviruses,
parvoviruses, papovaviruses (e.g., papillomaviruses), reoviruses
and adenoviruses) whose intact virions may be isolated by
techniques obvious to those skilled in the art (see Luria, S. E.,
et al. General Virology, 3rd ed., John Wiley & Sons, New York
p33-34 for general methods of virus purification, and relevant
chapters and references therein in Fields, B. N. et al.,
Fundamental Virology 3rd edition, Lippincott-Raven, New York 1996
for propagation and purification of specific virus groups), or for
any enveloped virus (including HIV, hepatitis B and herpes viruses)
which contain a stable nucleocapsid particle which can be isolated
by those skillful in the art (Luria, S. E. et al., supra; Fields,
B. N. et al., supra).
[0022] A viral site is used as a probe in the cell-free screen
assays of the present invention. The viral sites used in the assay
can be in their isolated form or contained within a virus or part
of a virus, e.g., viral capsid. The viral site can be any viral
region or binding pocket in any component of a virus that is
involved in viral infection, e.g., viral activation, replication,
and structure rearrangements. Any viral site for which compound
binding inhibits viral infection is contemplated in the present
invention. For example, the viral site may be those that constitute
the viral capsid or binding sites for known anti-viral
compounds.
[0023] Binding sites for anti-viral compounds are known and can be
easily obtained. For instance, crystallographic studies on various
strains of poliovirus and rhinovirus have shown that known
anti-picornaviral compounds bind to a conserved, hydrophobic pocket
at the core of VP1 which is one of four proteins constituting the
viral capsid. Tight binding of a therapeutic compound in this
pocket could prevent structural rearrangements in VP1 and the
subsequent transition of the mature virus into an infection
intermediate. Such a conserved hydrophobic pocket at the core of
viral capsid proteins, e.g., VP1, can be used as target site for
the screening assays of the present invention.
[0024] The initial phase of the screening assay involves the
incubation of viral sites with libraries of compounds to allow for
the specific interaction between the compounds and the viral sites.
The amount of virus and drug used in the incubation may be adjusted
in order to maximize the sensitivity or the specificity of the
assay. In general, the amount of virus and therefore the number of
sites will be limiting, and must be adjusted such that the total
number of sites are sufficient to guarantee detection of a bound
compound. The amount of compound used in the screening assay is
usually in the micromolar to nanomolar range. Preferably, it is
from about 1 .mu.M to about 400 .mu.M, more preferably from about
50 .mu.M to about 200 .mu.M, and most preferably from about 100
.mu.M to 200 .mu.M. The amount of viral binding sites used is
related to the amount of compounds, the recovery rate of the
complexes, and the limitation of the detection method used. The
ratio of the amount of compounds to viral sites can be varied from
about 500:1 to about 1:1, preferably from about 100:1 to about
10:1. The ratio of the concentration of compounds to viral sites
may be adjusted as needed to detect up to a specific number of
binders given the sensitivity of the detection system.
Alternatively the ratio of compounds to viral sites may be
increased in order to allow detection of binding in cases where all
compounds in a library bind weakly. The appropriate amounts of
virus and compound-to-virus ratios easily may be determined by one
skilled in the art.
[0025] The incubation of viral sites and compounds may be carried
out at room temperature for a few hours, e.g., one to four hours or
at 4.degree. C. for a longer period of time, e.g., overnight.
Different viral sites may require different incubation temperature
and incubation time. Such conditions can be easily determined by
one skilled in the art.
[0026] The viral site-bound compounds can be separated from unbound
compounds via any means known in the art. For example, the
compound-virus complexes may be isolated by loading the incubation
mixture onto a size exclusion column, e.g., a Sephacryl S-200 or
S-300 column, developing the column and selecting the fraction
containing virus as indicated by absorption at 280 nm. The bound
compounds may then be extracted from the isolated compound-virus
complexes via any means known in the art. For example, the
compounds may be extracted by differential solvent extraction of
the isolated complexes, for example by extraction with ethyl
acetate or any appropriate solvent which is not miscible in water.
Mass spectroscopy is usually used to detect and identify the
compounds which have been extracted from the compound-virus
complexes. Typically, electro-spray mass spectroscopy or matrix
assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectroscopy are used.
[0027] 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
Formation of Virus-Compound Complexes
[0028] 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. 1 for
schematic).
[0029] 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.
[0030] 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.
EXAMPLE 2
Purification of Virus-Compound Complexes
[0031] 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. 1 for schematic).
EXAMPLE 3
Extraction and Concentration of Virus-Bound Drug
[0032] 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.times.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. 1 for schematic).
EXAMPLE 4
Preparation of Sample for MS
[0033] 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.
EXAMPLE 5
1) Optimization of Assay Conditions with Radiolabeled Compounds
[0034] 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.
[0035] 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 5000 TD 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%.
[0036] 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.3H-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.
2) Assay Detects Binding of Drug with nM MIC
[0037] 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 VP1 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.
3) Assay Detects Binders in a Mixture of Compounds with MIC Values
Ranging From nM to .mu.M
[0038] 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.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. 2).
4) Ability of Assay to Discriminate Between Binders and
Non-Binders
[0039] 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.
5) Library A+Virus
[0040] A library of 75 potential ligands was synthesized using
combinatorial chemistry. 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.
[0041] 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
crystallographic).
[0042] 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.
6) A Hybrid Split-Pool Synthesis Approach
[0043] 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.
[0044] To test this approach, two small libraries containing six
compounds each, termed Libraries 6.1 and 6.2, respectively, were
synthesized (FIGS. 3 and 4) 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. 3 and 4). 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.
[0045] 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.
7) A Functional Assay for Screening Libraries
[0046] 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.
[0047] 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. 5) 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, radiolabeled 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.
[0048] 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-135S 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. 6). 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.
8) Crystallographic Assay Confirms Binding in Normal Drug Binding
Pocket
[0049] 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.
9) Stabilization of Virus is Entropically Determined
[0050] 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. 7). 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. 6), 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. Helps and Post, 1995,
J. mol. Biol. 254: 544, reached a similar conclusion from molecular
dynamics simulations of free rhinovirus and rhinovirus drug
complexes.
10) Infectivity Inhibition Assay
[0051] 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 Jensen was adapted for use in these screening approaches.
Briefly, serial five-fold dilutions of compounds are added directly
to a 96 well microliter plate. Approximately 100 TKT 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%.
[0052] As these preliminary experiments show, the technique
described above is effective in identifying new potential
anti-picornaviral compounds. The merging of computational drug
design methods and combinatorial chemistry allows for the design
and rapid synthesis of structure-based libraries of potential
ligands. The assay of the invention is a sensitive method, capable
of rapidly screening such libraries within hours. It is also
possible to extend this technique to screen for ligand binding in
other virus systems. This method may be generally used to screen
for binding to any nonenveloped virus (including picornaviruses,
calciviruses, paroviruses, papoviruses (e.g., papillomaviruses),
retroviruses and adenoviruses) whose intact virions may be isolated
by techniques obvious to those skilled in the art (Luria, S. E. et
al., supra; Fields, B. N. et al., supra), or for any enveloped
virus (including hepatitis B virus, alpha and flaviviruses, herpes
viruses, and retroviruses (including HIV)) which contain a stable
nucleocapsid particle which can be isolated by those skillful in
the art (Luria, S. E. et al., supra; Fields, B. N. et al., supra).
Once these potential leads have been identified by this preliminary
screen, they can be individually synthesized and subjected to a
battery of in vitro and in vivo tests to confirm their potency.
[0053] 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
* * * * *