Patent Application: US-69255603-A

Abstract:
the present invention relates to a method for selecting an anti - herpes viral compound and a method for selectively inhibiting herpesvrus in a human host in need of such treatment . the present invention relates to a method for selecting an anti - herpes viral compound and a method for selectively inhibiting herpesvrus in a human host in need of such treatment .

Description:
a key enzyme in the replication of all herpesviruses is the virus - coded dna polymerase . most of the currently available anti - herpes drugs target the viral dna polymerase . drugs such as foscarnet acts by direct inhibition of the viral polymerase . these drugs are non - nucleoside inhibitors of herpesvirus dna polymerases . others such as the nucleoside analogs , acyclovir , penciclovir and ganciclovir must first be phosphorylated to the monophosphate forms by virus encoded kinases and , further phosphorylated to triphosphate by cellular enzymes before they are active inhibitors . the triphosphate forms of these nucleoside analogs inhibit polymerases by competing with the binding of natural triphosphates and their subsequent insertion into growing dna strands . these drugs are known as nucleoside inhibitors of herpesvirus dna polymerases . one of the limitations of the currently available drugs is that they are active against only a few of the eight human herpesviruses . for example , acyclovir and penciclovir inhibit hsv and vzv replication but have poor activity against cmv . in order to identify antiviral compounds that would have the potential to inhibit replication of most of the human herpesviruses , compounds are in vitro screened for inhibitors of herpesvirus dna polymerase activity . because portions of the amino acid sequence of the polymerases are highly conserved within the herpesvirus family it is possible to discover small molecules that inhibit herpesvirus polymerases but not cellular dna polymerases . using this biochemical approach , several new classes of compounds such as the 4 - hydroxyquinoline derivatives ( 4 - hq ), 4 - oxo - dihydroquinoline derivatives ( 4 - oxo - dhq ) and 4 - oxo - dihydrothienopyridine derivatives ( 4 - oxo - dhtp ) were discovered as potent , non - nucleoside herpesvirus dna polymerase inhibitors . in vitro polymerase assays and / or in vivo cell culture assays have demonstrated that these compounds inhibit hsv - 1 , hsv - 2 , hcmv , vzv , ebv , and hhv - 8 replication . wherein ring a is a saturated or unsaturated fused double or triple heterocyclic ring having 1 , 2 , 3 or 4 heteroatoms selected from group consisting of oxygen , sulfur , or nitrogen ; and wherein r and x are the appropriated substitutents , respectively . examples of 4 - hq compounds , 4 - oxo - dhq compounds and 4 - oxo - dhtp compounds are illustrated in fig1 . antiviral activity of these examples are shown in table 1 below . as shown in table 1 , these compounds inhibit hsv - 1 and hsv - 2 as well or better than the current commercially available drug acyclovir . it has also been discovered that point mutations within the hsv - 1 polymerase gene that confer resistance to acyclovir and other nucleoside analogs do not result in resistance to the 4 - hq , 4 - oxo - dhqs or 4 - oxo - dhtps . serial passage of wild type hsv - 1 in the presence of 4 - oxo - dhq results in the isolation of mutants that are highly resistant (& gt ; 20 fold increase in the ic 50 ) to these compounds while retaining sensitivity to nucleoside inhibitors such as acyclovir . in order to determine the mechanism of action of 4 - hq , 4 - oxo - dhq and 4 - oxo - dhtp compounds against herpes viruses , mutants resistant to these compounds are isolated by serial passage of the virus in the presence of a 4 - oxo - dhq compound . sequencing analysis of hsv - 1 and hsv - 2 strains resistant to the 4 - oxo - dhq identifies that hsv - 1 ( kos strain ) polymerase protein and its homologous hsv - 2 have a conserved region ( a binding domain ), which is a critical contact point for these compounds . while amino acid numbering of the dna polymerase may vary between strains of hsv - 1 and hsv - 2 , this binding domain encompassing the hsv - 1 ( kos ) strain amino acid 823 is highly conserved in herpesviruses and can be identified by alligning the homologous amino acids of this domain as shown in fig2 . in hsv - 1 and hsv - 2 strains resistant to the 4 - oxo - dhq and similar compounds , a change of valine to an alanine at the binding domain provides full resistance . in the hsv - 1 dna polymerase , resistance is also found when a valine changes to methionine at amino acid 823 but only when accompanied by a second amino acid change . isolation of hcmv resistant to 4 - oxo - dhq &# 39 ; s is found to be very difficult . comparison of the amino acid sequence of the hsv polymerase ( y - g - f - t - g - v - q - h - g ) and hcmv polymerase ( y - g - f - t - g - v - v - n - g ) in the region of amino acid 823 ( underlined amino acid ) shows that there is a second valine at position 824 in the hcmv polymerase . in vitro assay using mutant hcmv polymerases demonstrates that full resistance to the 4 - oxo - dhqs requires changes at both amino acids 823 ( a valine to alanine ) and 824 ( a valine to leucine ). a hcmv polymerase gene containing v823a and v824l mutations is used in marker rescue experiments to generate a viral mutant . this mutant has an ic 50 approximately 7 - fold above that of wild - type hcmv . the hsv - 1 , hsv - 2 and hcmv mutants are also found to be resistant to other non - nucleoside inhibitors such as the 4 - oxo - dhtp and similar compounds . however , when the binding domain mutants ( e . g . hsv - 1 v823a , hsv - 2 - ms v826a , hsv - 2 - 186 v828a , and hcmv v823a / v824l mutants ) are tested in plaque reduction assays against a series of nucleoside polymerase inhibitors and the non - nucleoside inhibitor such as foscarnet , replication of the mutants is found to be inhibited by all of the currently marketed anti - herpes polymerase inhibitors tested . these studies demonstrate that certain non - nucleosides like 4 - hq , 4 - oxo - dhq and 4 - oxo - dhtp compounds bind to a different site on the herpes polymerase than the nucleoside inhibitors and foscarnet . the valine at the binding domain is conserved in the dna polymerases of six of the eight human herpesviruses and several animal herpesviruses , and appears to play a critical role in the antiviral activity of the 4 - hq , 4 - oxo - dhq and 4 - oxo - dhtp compounds . ( see fig2 ) since mutation at the binding domain negates these non - nucleoside inhibitors &# 39 ; activities , compounds could be tested against wild type polymerases and the mutant polymerases to establish the probability of similar binding . we refer to this property of compounds as interaction with the binding domain . since compounds that interact with the binding domain have exhibited broad - spectrum activity against herpesviruses , this invention provides a method for selecting compounds to treat individuals such as immunocompromised patients who are afflicted with multple herpesvirus infections . the term “ wild - type ” refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source . a wild - type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “ normal ” or “ wild - type ” form of the gene . in contrast , the term “ mutant ” refers to a gene or gene product which displays modifications in sequence and or functional properties ( i . e ., altered characteristics ) when compared to the wild - type gene or gene product . it is noted that naturally - occurring mutants can be isolated , these are identified by the fact that they have altered characteristics when compared to the wild - type gene or gene product . ic 50 refers to concentration of a drug that inhibits virus growth by 50 %. wild type hsv - 1 and hsv - 2 strains are listed in fig4 . the term “ binding domain ” refers to a conserved region in herpesvirus dna polymerases . the herpesvirus dna polymerases have seven ( 7 ) conserved regions . the binding domain is within the thrid conserved region ( see fig2 ). when the binding domain contacts with an inhibitor , at least one amino acid in the binding domain mutates and provides the resistance . in general , the binding domain is at an amino acid sequence position 818 - 829 of the hsv - 1 dna polymerase or the homologous region in other herpes virus dna polymerases ( see fig2 ). the term “ a binding domain mutant herpes virus ” refers to a herpes virus containing a binding domain mutation . more specifically , the binding domain in hsv - 1 strains , kos , f , djl and patton are at amino acid sequence position 823 . the binding domain in hsv - 2 ms - m1 strain is at amino acid sequence position 826 . the binding domain in hsv - 2 186 strain is at amino acid sequence position 828 . the binding domain in hcmv ad 169 strains is at amino acid sequence position 823 - 824 . the term “ xxxxy ” refers to an amino acid sequence position xxx , a single amino acid x in wild type is changed to an amino acid y . for example , the term “ v823a ” refers to an amino acid sequence position 823 , a valine found in wild type is changed to alanine in mutant strain . the term “ v824l ” refers to an amino acid sequence position 824 , a valine found in wild type is changed to leucine in mutant strain . the term “ v826a ” refers to an amino acid sequence position 826 , a valine found in wild type is change to alanine in mutant strain . the term “ v828a ” refers to an amino acid sequence position 828 , a valine found in wild type is change to alanine in mutant strain . a table of amino acids and their representative abbreviations , symbols and codons is set forth below in the following table . abb - sym - amino acid rev . bol codon ( s ) alanine ala a gca gcc gcg gcu cysteine cys c ugc ugu aspartic acid asp d gac gau glutamic acid glu e gaa gag phenylalanine phe f uuc uuu glycine gly g gga ggc ggg ggu histidine his h cac cau isoleucine ile i aua auc auu lysine lys k aaa aag leucine leu l uua uug cua cuc cug cuu methionine met m aug asparagine asn n aac aau proline pro p cca ccc ccg ccu glutamine gln q caa cag arginine arg r aga agg cga cgc cgg cgu serine ser s agc agu uca ucc ucg ucu threonine thr t aca acc acg acu valine val v gua guc gug guu tryptophan trp w ugg tyrosine tyr y uac uau african green monkey kidney cells ( vero ) and human foreskin fibroblast cells ( hff ) and herpes viruses can be obtained from the american type culture collection ( atcc ). media is defined as dulbecco &# 39 ; s modified eagle media ( dmem ) containing 10 % fetal bovine serum ( fbs ) and supplemented with antibiotics . cells are maintained in media at 37 ° c . in a humidified atmosphere of 5 % co 2 . hsv - 1 strains f , patton and djl , hsv - 2 strains ms , 35d and 186 , and hcmv strain ad169 are used in these studies . strain djl is a clinical isolate of hsv - 1 isolated in our lab from a primary oral lesion . measuring ic 50 of a compound of interest that inhibits heroes viruses preparation of virus stocks : hsv - 1 and hsv - 2 stocks are grown in vero cells . hcmv stocks are grown in hff cells . approximately 1 ml of media containing sufficient virus to infect approximately 0 . 1 % to 1 % of the cells ( multiplicity of infection of 0 . 001 to 0 . 01 pfu / cell ) is added to a t - 150 cell culture flask containing a confluent monolayer of cells . the cells are incubated at 37 ° c . for approximately 1 hour . approximately 50 ml of media is then added to the flask and the cells are incubated at 37 ° c . until viral cytopathic effect ( cpe ) is apparent in 100 % of the cells . the flask is then placed at − 80 ° c . for at least 30 min . the flask containing frozen media and cells is placed in a 37 ° c . water bath until the media is thawed . this process disrupts the cells and releases virus into the media . 1 ml aliquots of media containing virus are dispensed into tubes and stored at − 80 ° c . these aliquots of media containing virus are referred to as virus stocks . titrating virus stocks : aliquots of virus are thawed at 37 ° c . and serially diluted ( 10 fold dilutions ) in media . 0 . 1 ml of each dilution of virus is placed in a single well of 24 - well cell culture dish containing a confluent monolayer of cells ( vero cells for hsv - 1 and hsv - 2 , hff cells for hcmv ) and incubated at 37 ° c . for 1 h . the virus innoculum is then removed and 1 ml of media containing 0 . 8 % carboxymethylcellulose ( cmc ) is added to each well of the dish . the dish is incubated at 37 ° c . for approximately 2 - 3 days ( hsv - 1 and hsv - 2 ) or 6 - 9 days ( hcmv ) to allow sufficient growth of virus to form plaques in the cell monolayer . plaques can be observed and counted microscopically or by staining the cells with 0 . 1 % crystal violet in 20 % ethanol . the virus titer which is expressed as plaque forming units ( pfu ) per ml is obtained by counting the plaques in a well and correcting for the dilution of the viral innoculum . plaque reduction assays : antiviral activity of compounds against herpesviruses such as hsv - 1 , hsv - 2 , or hcmv can be measured using plaque reduction assays . 0 . 1 ml of media containing approximately 50 pfu of virus is added to each well of a 24 - well cell culture dish containing a confluent monolayer of cells ( vero cells for hsv - 1 and hsv - 2 , hff cells for hcmv ). compounds are dissolved in 100 % dmso and diluted in 100 % dmso as 200 × stocks of the desired final drug concentration . typically 5 - 6 two - fold dilutions are prepared for each compound . dilutions of compounds are then added to media containing 0 . 8 % cmc resulting in a final 1 × drug concentration . after the virus - infected cells have incubated for 1 h at 37 ° c ., the virus innoculum is removed and 1 ml of media containing 0 . 8 % cmc and the various concentrations of compound is added to each well of the dish . the dish is incubated at 37 ° c . for approximately 2 - 3 days ( hsv - 1 and hsv - 2 ) or 6 - 9 days ( hcmv ) to allow sufficient growth of virus to form plaques in the cell monolayer . plaques can be observed and counted microscopically or by staining the cells with 0 . 1 % crystal violet in 20 % ethanol . virus inhibition is determined for each drug concentration by comparing the number of plaques in drug - containing wells to control wells that did not contain drug . antiviral activity of a compound is expressed as the concentration of compound predicted to reduce the number of plaques in a well by 50 % ( ic 50 ). the ic 50 values are calculated by plotting the percent inhibition vs . concentration of compound using excel software for linear regression . vero cells are plated out at a density of 3 . 5 × 10 5 cells per well in a six well tissue culture plate . cells are infected with hsv - 1 kos at a multiplicity of infection ( moi ) of 0 . 1 pfu / cell and 1 h post infection the cells are overlayed with 3 ml media containing 20 um of a 4 - oxo - dhq . cultures are incubated for 20 h at 37 ° c ., freeze / thawed to release cell - associated virus , and 0 . 1 ml of culture is used to infect a new monolayer of vero cells ( one passage ). serial passage is repeated seven times in the presence of 20 um drug . virus isolates are then plaque purified three times prior to preparation of stocks . virus recovered from each passage in the presence of compound no . 17 is shown in fig3 . 4 - oxo - dhq resistant hsv - 1 and hsv - 2 may also be selected by the marker transfer method described below using wild - type hsv dna and the corresponding mutant hsv polymerase gene . a plasmid containing the wild - type hcmv polymerase gene is modified to contain the v823a or v823a and v824l mutations using a site - directed mutagenesis kit ( stratagene corp .) and following the manufactures &# 39 ; s protocol . hff cells are plated into t25 tissue culture flasks to achieve 80 % confluency at the time of the transfection . wild type hcmv ad169 dna and plasmid dna containing the mutant hcmv polymerase gene are mixed at a ratio of 1 : 2 ( 2 ug of viral dna to 4 ug of plasmid dna ). dna &# 39 ; s are transfected using superfect transfection reagent according to methods recommended by the manufacturer ( quiagen inc .). cells are harvested five days posttransfection , freeze - thawed to release virus and half of the sample is used to infect 1ff cell monolayers . cells are overlayed with media containing 20 um 4 - oxo - dhq compound 2 ( see fig1 ). serial passage is repeated seven times in the presence of 20 um compound 2 and virus isolates are then plaque purified three times prior to preparation of viral stock . hsv dna is purified from the cytoplasm of infected vero cells . vero cells ( 50 % confluent ) are infected at an multiplicity of 0 . 01 pfu / cell . at 3 - 5 days postinfection infected cells ( 100 % cpe ) are harvested by centrifugation at 1000 rpm in a beckman gs - 6r centrifuge . the pelleted cells are resuspended in te buffer and placed on ice for 15 minutes . np - 40 is then added to a final concentration of 0 . 2 % and incubated on ice for a further 15 minutes . the cells are centrifuged at 2000 rpm for 10 minutes in a beckman gs - 6r centrifuge . the supernatant is removed and edta is added to a final concentration of 20 mm followed by the addition of sds to a final concentration of 0 . 3 % and proteinase k to a concentration of 50 ug / ml then incubated at 45 c for 2 hours . hcmv dna is isolated by infecting hff cells ( 25 % confluency ) with hcmv at an multiplicity of 0 . 1 pfu / cell . cells and media are harvested 5 - 7 days postinfection ( 100 % cpe ) and subjected to low speed centrifugation to remove intact cells and cell debris followed by a high speed spin to pellet virus particles ( 2500 rpm &# 39 ; s in a beckman sw28 rotor for 1 hour ). following incubation of the hsv and hcmv samples , 1 . 5 volumes of saturated nai is added to the digested extract and the refractive index is adjusted to 1 . 434 - 1 . 435 . ethidium bromide is added to a final concentration of 50 ug / ml . the samples are loaded into a vti 50 centrifuge tube and spun for 24 hours at 45 , 000 rpm . the dna band is harvested extracted three times with n - butanol , then dialyzed against te buffer followed by a dialysis against 95 % ethanol and a final dialysis against te buffer . hsv - 1 , hsv - 2 or hcmv viral dna &# 39 ; s are sequenced directly using an abi377 fluorescence sequencer ( perkin elmer applied biosystems , foster city , calif .) and the abi bigdye prism ™ drhodamine terminator cycle sequencing ready reaction kit with amplitaq fs ™ dna polymerase ( pe applied biosystems ). each cycle sequencing reaction contained about 1 . 0 ug of purified viral dna . cycle - sequencing is performed using an initial denaturation at 98 ° c . for 1 min , followed by 50 cycles : 98 ° c . for 30 sec , annealing at 50 ° c . for 30 sec , and extension at 60 ° c . for 4 min . temperature cycles and times are controlled by a perkin - elmer 9700 thermocycler . extension products are purified using centriflex ™ gel filtration cartridges ( edge biosystems , gaithersburg , md .). each reaction product is loaded by pipette onto the column , which is then centrifuged in a swinging bucket centrifuge ( sorvall model rt6000b table top centrifuge ) at 750 × g for 1 . 5 min at room temperature . column - purified samples are dried under vacuum for about 40 min and then dissolved in 4 ul of a dna loading solution ( 83 % deionized formamide , 8 . 3 mm edta , and 1 . 6 mg / ml blue dextran ). the samples are then heated to 90 ° c . for two min , and held at 4 ° c . until loading . 1 . 5 ul of each sample is loaded into a single well of the abi377 sequencer . sequence chromatogram data files from the abi377 are analyzed with the computer program sequencher ( gene codes , ann arbor , mich . ), for assembly of sequence fragments and correction of ambiguous base calls . generally sequence reads of 600 - 700 bp are obtained . potential sequencing errors are minimized by obtaining sequence information from both dna strands and by re - sequencing difficult areas using primers at different locations until all sequencing ambiguities are removed . the entire coding region of the polymerase genes from both the parent strains and the resistant viruses are sequenced . the dna sequencing is done using viral dna as the template thus avoiding cloning of the polymerase genes . the amino acid sequence of the dna polymerases of hsv - 1 kos , f , patton and djl and hsv - 2 ms and 186 are compared in fig4 . amino acids that are identical for the six polymerases are shaded in black while regions where amino acid differences are found are shaded in gray . the amino acid sequence of the four hsv - 1 polymerases are essentially identical with only a few minor changes noted between the different hsv - 1 strains . the majority of amino acid changes are found when the sequences of the hsv - 1 and hsv - 2 polymerases are compared . isolation and characterization of hsv - 1 and hsv - 2 mutants that are resistant to the 4 - oxo - dho &# 39 ; s and 4 - oxo - dhtp compounds a panel of viruses consisting of four strains of hsv - 1 ( kos , f , djl , patton ) and three strains of hsv - 2 ( ms , 35d , 186 ) are tested in a plaque reduction assay against four different 4 - oxo - dhq compounds (# 1 , 2 , 4 , 5 as shown in fig1 ), and one 4 - oxo - dhtp compound (# 3 as shown in fig1 ) and against acyclovir . the six drugs inhibited replication of the seven virus strains with ic 50 values ranging from 2 - 10 μm ( table 1 ). in order to select for 4 - oxo - dhq resistant mutants , hsv - 1 strains kos , f , and djl along with hsv - 2 strains 186 and ms are serially passaged in the presence of 20 um compound 1 . following the seventh passage , 4 - oxo - dhq resistant virus from each strain are plaque purified three times and high - titer stocks are made . all of the resistant hsv mutants grew to high titers in vero cells , indicating that the mutations in the resistant isolates did not significantly impair their growth . the mutants selected with 4 - oxo - dhq compound 1 exhibited & gt ; 10 fold increase in ic 50 when tested in a plaque reduction assay against 4 - oxo - dhq compound 1 data are shown in table 2 . dna sequence analysis of the 4 - oxo - dhq resistant mutants ( hsv - 1 kos - m1 , hsv - 1 f - m1 , hsv - 1 djl - m1 , hsv - 2 186 - m1 , hsv - 2 ms - m1 ) demonstrated that all five mutants contained a single point mutation of t to c at the binding domain resulting in a valine to alanine amino acid change . isolation and characterization of a hcmv mutant that is resistant to the 4 - oxo - dho &# 39 ; s and 4 - oxo - dhtp compounds in order to select for a 4 - oxo - dhq hcmv resistant mutant , virus ( strain ad169 ) is serially passaged in the presence of 20 um a 4 - oxo - dhq . although we could readily select for hsv mutants using this procedure we failed to isolate an hcmv mutant , even when the virus is passaged at low drug concentrations (& lt ; 5 um ). comparison of the amino acid sequence of the hsv polymerase , y - g - f - t - g - v - q - h - g , and hcmv polymerase , y - g - f - t - g - v - v - n - g , in the region of amino acid 823 ( underlined amino acid ) showed that there is a second valine at position 824 in the hcmv polymerase . in order to determine if both valines need to be changed in order to confer resistance to the 4 - oxo - dhq &# 39 ; s , in vitro polymerase assays are done using mutant hcmv polymerases containing either v823a or v823a plus v824l ( table 3 ). the v823a alone resulted in a 3 . 5 - fold increase in the ic 50 while the polymerase with the double amino acid change had nearly 10 - fold increase in the ic 50 . in order to isolate an hcmv resistant mutant marker rescue experiments are done . plasmids containing the mutant polymerase genes are transfected into hff cells along with wild type hcmv ad169 dna . the resulting virus is then serially passaged in the presence of 20 um compound 1 ( see fig1 ). a 4 - oxo - dhq resistant virus is isolated from marker rescue studies done with the hcmv polymerase gene containing mutations that result in the v823a , v824l amino acid changes , but not with the gene containing v823a change alone . the mutant selected with compound 1 ( hcmv ad169 - m1 ) exhibited ˜ 7 - fold increase in ic 50 when tested in a plaque reduction assay compared to ganciclovir and cidofovir which has a ≦ 2 - fold change in sensitivity ( table 4 ). the entire coding region of the hcmv polymerase genes from both the parent strain and the resistant virus are sequenced . the dna sequencing is again done using viral dna as the template thus avoiding cloning of the polymerase genes . comparison of the dna sequence of the two polymerase genes demonstrated that the resistant mutant contained two point mutations that resulted in the predicted v823a , v824l amino acid changes . as with the hsv resistant viruses these results demonstrate the critical role of the region encompassing amino acid 823 for inhibition of polymerase activity by these compounds . antiviral activity of nucleoside and non - nucleoside polymerase inhibitors against 4 - oxo - dho resistant mutants in order to determine if the 4 - hq binding domain mutations alter the sensitivity of the hsv - 1 , hsv - 2 and hcmv mutants to both non - nucleoside ( 4 - oxo - dhq &# 39 ; s ) and nucleoside inhibitors ( e . g acyclovir and ganciclovir ) several of the mutants are tested in plaque reduction assays against a series of non - nucleoside compounds including foscarnet ( pfa ), 4 - hq &# 39 ; s 4 - oxo - dhq &# 39 ; s and 4 - oxo - dhtp &# 39 ; s ( table 5 ). the mutants are also tested against a series of nucleoside inhibitors including acyclovir and ganciclovir ( table 5 ). the activity of these compounds against the mutants is compared to their activity against the wild type strains that are used to isolate the hsv and hcmv mutants . when tested against a number of 4 - hq &# 39 ; s , 4 - oxo - dhq &# 39 ; s and 4 - oxo - dhtp &# 39 ; s and other related classes of compounds all of the drugs are found to inhibit the wild type virus with ic 50 values ranging from & lt ; 0 . 1 um to 30 um . when these drugs are tested against the resistant viruses they are found to have ic 50 values 5 to 10 fold higher then the parent virus . there is little if any difference in the ic 50 values of the nucleoside compounds and the non - nucleoside pfa between the wild type and mutant hsv - 1 , hsv - 2 , and hcmv viruses . these results demonstrate that the amino acid change in the binding domain ( v823a in the hsv - 1 polymerase , v826a in the hsv2 - ms polymerase , v828a in the hsv2 - 186 polymerase , and the v823a / v824l changes in the hcmv polymerase ) resulted in resistance to the 4 - oxo - dhq &# 39 ; s and 4 - oxo - dhtp &# 39 ; s , which provides further evidence that these classes of compounds share an affinity for a region we refer to as the binding domain . in contrast , these amino acid changes did not alter the activity of these viruses to other classes of polymerase inhibitors . antiviral compounds identified by the present invention can conveniently be administered in a pharmaceutical composition containing the compound in combination with a suitable excipient , the composition being useful in combating viral infections . pharmaceutical compositions containing a compound appropriate for antiviral use are prepared by methods and contain excipients which are well known in the art . a generally recognized compendium of such methods and ingredients is remington &# 39 ; s pharmaceutical sciences by e . w . martin ( mark publ . co ., 15th ed ., 1975 ). antiviral compounds identified by the present invention and their compositions can by administered parenterally ( for example , by intravenous , intraperitoneal or intramuscular injection ), topically , orally , or rectally , depending on whether the preparation is used to treat internal or external viral infections . for oral therapeutic administration , the active compound may be combined with one or more excipients and used in the form of ingestible tablets , buccal tablets , troches , capsules , elixirs , suspensions , syrups , wafers , and the like . such compositions and preparations should contain at least 0 . 1 % of active compound . the percentage of the compositions and preparations may , of course , be varied and may conveniently be between about 2 to about 60 % of the weight of a given unit dosage form . the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained . the tablets , troches , pills , capsules , and the like may also contain the following : binders such as gum tragacanth , acacia , corn starch or gelatin ; excipients such as dicalcium phosphate ; a disintegrating agent such as corn starch , potato starch , alginic acid and the like ; a lubricant such as magnesium stearate ; and a sweetening agent such as sucrose , fructose , lactose or aspartame or a flavoring agent such as peppermint , oil of wintergreen , or cherry flavoring may be added . when the unit dosage form is a capsule , it may contain , in addition to materials of the above type , a liquid carrier , such as a vegetable oil or a polyethylene glycol . various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form . for instance , tablets , pills , or capsules may be coated with gelatin , wax , shellac or sugar and the like . a syrup or elixir may contain the active compound , sucrose or fructose as a sweetening agent , methyl and propylparabens as preservatives , a dye and flavoring such as cherry or orange flavor . of course , any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non - toxic in the amounts employed . in addition , the active compound may be incorporated into sustained - release preparations and devices . antiviral compounds identified by the present invention and their compositions can also be administered intravenously or intraperitoneally by infusion or injection . solutions of the active compound or its salts can be prepared in water , optionally mixed with a nontoxic surfactant . dispersions can also be prepared in glycerol , liquid polyethylene glycols , triacetin , and mixtures thereof and in oils . under ordinary conditions of storage and use , these preparations contain a preservative to prevent the growth of microorganisms . pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions , optionally encapsulated in liposomes . in all cases , the ultimate dosage form should be sterile , fluid and stable under the conditions of manufacture and storage . the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising , for example , water , ethanol , a polyol ( for example , glycerol , propylene glycol , liquid polyethylene glycols , and the like ), vegetable oils , nontoxic glyceryl esters , and suitable mixtures thereof . the proper fluidity can be maintained , for example , by the formation of liposomes , by the maintenance of the required particle size in the case of dispersions or by the use of surfactants . the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents , for example , parabens , chlorobutanol , phenol , sorbic acid , thimerosal , and the like . in many cases , it will be preferable to include isotonic agents , for example , sugars , buffers or sodium chloride . prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption , for example , aluminum monostearate and gelatin . sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above , as required , followed by filter sterilization . in the case of sterile powders for the preparation of sterile injectable solutions , the preferred methods of preparation are vacuum drying and the freeze drying techniques , which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile - filtered solutions . for topical administration , the present compounds may be applied in pure form , i . e ., when they are liquids . however , it will generally be desirable to administer them to the skin as compositions or formulations , in combination with a dermatologically acceptable carrier , which may be a solid or a liquid . useful solid carriers include finely divided solids such as talc , clay , microcrystalline cellulose , silica , alumina and the like . useful liquid carriers include water , alcohols or glycols or water - alcohol / glycol blends , in which the present compounds can be dissolved or dispersed at effective levels , optionally with the aid of non - toxic surfactants . adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use . the resultant liquid compositions can be applied from absorbent pads , used to impregnate bandages and other dressings , or sprayed onto the affected area using pump - type or aerosol sprayers . thickeners such as synthetic polymers , fatty acids , fatty acid salts and esters , fatty alcohols , modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes , gels , ointments , soaps , and the like , for application directly to the skin of the user . examples of useful dermatological compositions which can be used to deliver the compounds of formula i to the skin are known to the art ; for example , see jacquet et al . ( u . s . pat . no . 4 , 608 , 392 ), geria ( u . s . pat . no . 4 , 992 , 478 ), smith et al . ( u . s . pat . no . 4 , 559 , 157 ) and wortzman ( u . s . pat . no . 4 , 820 , 508 ). useful dosages of the compounds of formula i can be determined by comparing their in vitro activity , and in vivo activity in animal models . methods for the extrapolation of effective dosages in mice , and other animals , to humans are known to the art ; for example , see u . s . pat . no . 4 , 938 , 949 . the compound is conveniently administered in unit dosage form ; for example , containing 5 to 1000 mg , conveniently 10 to 750 mg , most conveniently , 50 to 500 mg of active ingredient per unit dosage form . the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals , for example , as two , three , four or more sub - doses per day . the sub - dose itself may be further divided , e . g ., into a number of discrete loosely spaced administrations ; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye . for internal infections , the compositions can be administered orally or parenterally at dose levels , calculated as the free base , of about 0 . 1 to 300 mg / kg , preferably 1 . 0 to 30 mg / kg of mammal body weight , and can be used in man in a unit dosage form , administered one to four times daily in the amount of 1 to 1000 mg per unit dose . for parenteral administration or for administration as drops , as for eye infections , the compounds are presented in aqueous solution in a concentration of from about 0 . 1 to about 10 %, more preferably about 0 . 1 to about 7 %. the solution may contain other ingredients , such as emulsifiers , antioxidants or buffers . generally , the concentration of the compound ( s ) of formula i in a liquid composition , such as a lotion , will be from about 0 . 1 - 25 wt -%, preferably from about 0 . 5 - 10 wt -%. the concentration in a semi - solid or solid composition such as a gel or a powder will be about 0 . 1 - 5 wt -%, preferably about 0 . 5 - 2 . 5 wt -%. the exact regimen for administration of the compounds and compositions disclosed herein will necessarily be dependent upon the needs of the individual subject being treated , the type of treatment and , of course , the judgment of the attending practitioner . the antiviral activity of a compound of the invention can be determined using pharmacological models which are well known to the art , or using test a described below . the compounds of formula ( i ) and pharmaceutically acceptable salts thereof are useful as antiviral agents . thus , they are useful to combat viral infections in animals , including man . the compounds are generally active against herpes viruses , and are particularly useful against the varicella zoster virus , the epstein - barr virus , the herpes simplex virus , the human herpes virus type 8 ( hhv - 8 ) and the cytomegalovirus ( cmv ). arg his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg ser leu asp glu asp ala pro ala glu gln arg thr gly val his asp gly arg leu arg arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly ala asp his ala pro lys gly phe asp pro thr tyr ser met arg ala ala gln leu his glu arg phe met asp ala ile asn lys ala glu val asp arg his leu gln cys arg ala pro arg asp phe val arg ser gly arg ala leu ala tyr leu cys asp asn phe cys pro ala ile arg lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys gly thr ser ser asp val glu phe asn cys thr ala asp asn leu ala val glu gly ala met cys asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala glu arg pro glu asp leu val ile gln ile ser cys leu leu tyr leu ala phe met thr phe val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe val leu thr lys leu thr glu ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys glu tyr cys val gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile cys leu leu arg leu ala gly gln lys gly phe ile leu pro asp thr phe ala ser leu tyr pro ser ile ile gln ala his asn leu cys phe lys ala his val arg glu ser leu leu ser ile leu leu arg asp trp cys asn ser val tyr gly phe thr gly ala gln his gly leu leu pro cys leu his val ala ala thr val thr thr ile gly arg glu met leu leu ala thr arg ala tyr val his ala arg trp ala glu phe asp gln tyr ser met arg ile ile tyr gly asp thr asp ser ile phe val leu val arg lys asn asn cys ala phe ile asn arg thr ser arg ala leu ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser arg his pro arg ala tyr thr asn lys arg leu ala his leu thr val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr glu leu ala glu asp pro gly tyr ala ile ala arg gly val pro leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu thr trp his pro pro asp arg his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg ser leu asp glu asp ala pro ala glu gln arg thr gly val his asp gly arg leu arg arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly ala asp his ala pro glu gly phe asp pro thr tyr ser met arg ala ala gln leu his glu arg phe met asp ala ile asn lys ala glu val asp arg his leu gln cys arg ala pro arg asp phe val arg ser gly arg ala leu ala tyr leu cys asp asn phe cys pro ala ile arg lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys gly thr ser ser asp val glu phe asn cys thr ala asp asn leu ala val glu gly ala met cys asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala glu arg pro glu asp leu val ile gln ile ser cys leu leu tyr leu ala phe met thr phe val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe val leu thr lys leu thr glu ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys glu tyr cys val gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile cys leu leu arg leu ala gly gln lys gly phe ile leu pro asp thr phe asp phe ala ser leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg pro glu ala val ala his leu glu asp trp leu ala met arg lys gln ile arg ser arg ile pro gln ser val val cys asn ser val tyr gly phe thr gly ala gln his gly leu leu pro cys leu his val ala ala thr val thr thr ile gly arg glu met leu leu ala thr arg ala tyr val his ala arg trp ala glu phe asp leu val arg lys asn asn cys ala phe ile asn arg thr ser arg pro glu gly leu gln ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser arg his pro arg ala tyr thr asn lys arg gln val pro ser ile lys asp arg ile pro tyr val ile val ala val ser glu leu ala glu asp pro gly tyr ala ile ala arg gly val pro leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu thr trp his pro his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr val tyr gly phe thr gly ala gln his gly leu leu pro cys leu his arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val ala his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr val tyr gly phe thr gly ala gln his gly leu leu pro cys leu his arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val ala his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr val tyr gly phe thr gly ala gln his gly leu leu pro cys leu his arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val ala met phe phe asn pro tyr leu ser gly gly val thr gly gly ala val val met phe asp gly gln thr gly leu ile lys his lys thr gly arg leu pro leu met phe tyr arg glu ile lys his leu leu ser his asp gly pro ile arg phe his thr tyr asp gln thr asp ala val leu phe phe asp ser pro glu asn val ser pro arg tyr arg gln his leu val pro ser gly asn val leu arg phe phe gly ala thr glu his gly tyr ser ile cys val asn val phe gly gln arg ser tyr phe tyr cys glu tyr ser asp thr asp arg leu arg glu val ile ala ser val gly glu thr lys thr ser ile tyr gly tyr gly thr arg pro val pro asp leu gln cys val ser ile ser asn trp thr met ala arg lys ile gly glu ser val asn arg tyr asp trp arg gln gln gly arg ala ser thr cys asp ser ser trp pro arg tyr arg cys leu ser phe asp ile glu cys met ser gly glu gly gly phe pro cys ala glu lys ser asp asp ile val ile gln ile ser cys val cys tyr glu thr gly gly asn thr ala val asp gln gly ile pro asn gly asn asp gly arg gly cys thr ser thr cys gly gln val gly pro asp val asp val tyr glu phe pro ser pro ala phe val thr gly tyr asn ile asn ser phe asp leu lys tyr ile leu thr arg leu glu tyr leu tyr lys val asp ser gln arg phe cys lys leu pro thr ala gln gly gly arg phe phe leu his ser pro his asn asn pro ala ser thr ala ala thr lys val tyr ile ala gly ser val val ile asp met tyr pro val cys met ala lys thr asn ser pro asn tyr lys leu asn thr met ala glu leu tyr leu arg gln arg lys asp asp leu ser tyr lys asp ile pro arg cys phe val ala asn leu val arg asp leu phe asn thr ile asn phe his tyr glu ala gly gly gln gln ile arg ile tyr thr ser leu leu asp glu cys ala cys arg asp phe ile leu pro asn his tyr ser lys gly thr thr val pro leu tyr pro ser ile ile met ala his asn leu cys tyr ser thr leu val thr leu glu asn gly val thr his arg phe val arg ala ser val met leu leu asp lys glu gln met ala leu lys val thr cys asn ala ala arg phe ile lys asp asn phe ser glu pro cys phe leu his asn phe phe asn gln glu asp tyr val val gly thr arg glu gly asp ser ser asn glu arg arg val glu ala arg val ile tyr gly asp thr asp arg gly pro ser leu ala his tyr val thr ala cys leu phe val glu cys lys lys arg tyr ile gly lys val glu gly ala ser gly leu ser met lys gly val asp leu val arg lys thr ala cys glu phe val lys lys lys tyr gly val pro arg gly phe trp arg ile leu arg arg tyr arg gln ser asn leu pro his ile ala val ile lys arg leu ala ala arg ser glu glu leu pro ser val gly asp arg val phe tyr val leu thr ala pro gly val arg thr ala pro gln gly ser arg lys pro pro ser ala val cys asn tyr glu val ala glu asp pro ser tyr val arg glu his gly val pro ile his ala asp lys tyr phe glu gln val leu lys ala val thr asn val leu ser pro val phe pro gly gly glu thr ala arg lys asp lys phe leu his met phe phe asn pro tyr leu ser gly gly val thr gly gly ala val val met phe asp gly gln thr gly leu ile lys his lys thr gly arg leu pro leu met phe tyr arg glu ile lys his leu leu ser his asp gly pro ile arg phe his thr tyr asp gln thr asp ala val leu phe phe asp ser pro glu asn val ser pro arg tyr arg gln his leu val pro ser gly asn val leu arg phe phe gly ala thr glu his gly tyr ser ile cys val asn val phe gly gln arg ser tyr phe tyr cys glu tyr ser asp thr asp arg leu arg glu val ile ala ser val gly glu thr lys thr ser ile tyr gly tyr gly thr arg pro val pro asp leu gln cys val ser ile ser asn trp thr met ala arg lys ile gly glu ser val asn arg tyr asp trp arg gln gln gly arg ala ser thr cys asp ser ser trp pro arg tyr arg cys leu ser phe asp ile glu cys met ser gly glu gly gly phe pro cys ala glu lys ser asp asp ile val ile gln ile ser cys val cys tyr glu thr gly gly asn thr ala val asp gln gly ile pro asn gly asn asp gly arg gly cys thr ser thr cys gly gln val gly pro asp val asp val tyr glu phe pro ser pro ala phe val thr gly tyr asn ile asn ser phe asp leu lys tyr ile leu thr arg leu glu tyr leu tyr lys val asp ser gln arg phe cys lys leu pro thr ala gln gly gly arg phe phe leu his ser pro his asn asn pro ala ser thr ala ala thr lys val tyr ile ala gly ser val val ile asp met tyr pro val cys met ala lys thr asn ser pro asn tyr lys leu asn thr met ala glu leu tyr leu arg gln arg lys asp asp leu ser tyr lys asp ile pro arg cys phe val ala asn leu val arg asp leu phe asn thr ile asn phe his tyr glu ala gly gly gln gln ile arg ile tyr thr ser leu leu asp glu cys ala cys arg asp phe ile leu pro asn his tyr ser lys gly thr thr val pro leu tyr pro ser ile ile met ala his asn leu cys tyr ser thr leu val thr leu glu asn gly val thr his arg phe val arg ala ser val met leu leu asp lys glu gln met ala leu lys val thr cys asn ala ala arg phe ile lys asp asn phe ser glu pro cys phe leu his asn phe phe asn gln glu asp tyr val val gly thr arg glu gly asp ser ser asn glu arg arg val glu ala arg val ile tyr gly asp thr asp arg gly pro ser leu ala his tyr val thr ala cys leu phe val glu cys lys lys arg tyr ile gly lys val glu gly ala ser gly leu ser met lys gly val asp leu val arg lys thr ala cys glu phe val lys lys lys tyr gly val pro arg gly phe trp arg ile leu arg arg tyr arg gln ser asn leu pro his ile ala val ile lys arg leu ala ala arg ser glu glu leu pro ser val gly asp arg val phe tyr val leu thr ala pro gly val arg thr ala pro gln gly ser arg lys pro pro ser ala val cys asn tyr glu val ala glu asp pro ser tyr val arg glu his gly val pro ile his ala asp lys tyr phe glu gln val leu lys ala val thr asn val leu ser pro val phe pro gly gly glu thr ala arg lys asp lys phe leu his arg his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg ser leu asp glu asp ala pro ala glu gln arg thr gly val his asp gly arg leu arg arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly ala asp his ala pro lys gly phe asp pro thr tyr ser met arg ala ala gln leu his glu arg phe met asp ala ile asn lys ala glu val asp arg his leu gln cys arg ala pro arg asp phe val arg ser gly arg ala leu ala tyr leu cys asp asn phe cys pro ala ile arg lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys gly thr ser ser asp val glu phe asn cys thr ala asp asn leu ala val glu gly ala met cys asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala glu arg pro glu asp leu val ile gln ile ser cys leu leu tyr leu ala phe met thr phe val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe val leu thr lys leu thr glu ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys glu tyr cys val gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile cys leu leu arg leu ala gly gln lys gly phe ile leu pro asp thr phe ala ser leu tyr pro ser ile ile gln ala his asn leu cys phe lys ala his val arg glu ser leu leu ser ile leu leu arg asp trp cys asn ser val tyr gly phe thr gly val gln his gly leu leu pro cys leu his val ala ala thr val thr thr ile gly arg glu met leu leu ala thr arg ala tyr val his ala arg trp ala glu phe asp gln tyr ser met arg ile ile tyr gly asp thr asp ser ile phe val leu val arg lys asn asn cys ala phe ile asn arg thr ser arg ala leu ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser arg his pro arg ala tyr thr asn lys arg leu ala his leu thr val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr glu leu ala glu asp pro gly tyr ala ile ala arg gly val pro leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu thr trp his pro pro asp arg his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg ser leu asp glu asp ala pro ala glu gln arg thr gly val his asp gly arg leu arg arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly ala asp his ala pro glu gly phe asp pro thr tyr ser met arg ala ala gln leu his glu arg phe met asp ala ile asn lys ala glu val asp arg his leu gln cys arg ala pro arg asp phe val arg ser gly arg ala leu ala tyr leu cys asp asn phe cys pro ala ile arg lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys gly thr ser ser asp val glu phe asn cys thr ala asp asn leu ala val glu gly ala met cys asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala glu arg pro glu asp leu val ile gln ile ser cys leu leu tyr leu ala phe met thr phe val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe val leu thr lys leu thr glu ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys glu tyr cys val gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile cys leu leu arg leu ala gly gln lys gly phe ile leu pro asp thr phe asp phe ala ser leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg pro glu ala val ala his leu glu asp trp leu ala met arg lys gln ile arg ser arg ile pro gln ser val val cys asn ser val tyr gly phe thr gly val gln his gly leu leu pro cys leu his val ala ala thr val thr thr ile gly arg glu met leu leu ala thr arg ala tyr val his ala arg trp ala glu phe asp leu val arg lys asn asn cys ala phe ile asn arg thr ser arg pro glu gly leu gln ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser arg his pro arg ala tyr thr asn lys arg gln val pro ser ile lys asp arg ile pro tyr val ile val ala val ser glu leu ala glu asp pro gly tyr ala ile ala arg gly val pro leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu thr trp his pro his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala 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gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val thr his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val ala his thr tyr tyr ser glu cys asp glu phe arg phe ile ala pro arg gly his leu lys arg ala pro lys val tyr cys gly gly asp glu arg leu trp gly gly val asp his ala pro ala gly phe asn pro thr val thr val phe his val tyr asp ile leu glu asn val glu his ala tyr arg val ala val his val tyr gly thr arg gln tyr phe tyr met asn lys glu glu val asp arg his leu gln cys arg ala pro arg asp leu arg gly ile ser ala asp his phe glu ala glu val val glu arg thr val arg ser gly arg val leu ser tyr leu cys asp asn phe cys pro ala ile lys lys tyr glu gly gly val asp ala thr thr arg phe ile leu asp asn pro gly phe val thr phe gly trp tyr arg leu lys pro thr ser ser asp val glu phe asn cys thr ala asp asn leu ala ile glu gly gly met ser asp leu pro ala tyr lys leu met cys phe asp ile glu cys lys ala gly gly glu asp glu leu ala phe pro val ala gly his pro glu asp leu val ile gln ile ser cys leu leu tyr asp cys asp leu pro glu ser his leu asn glu leu ala ala arg gly leu ala phe met thr leu val lys gln tyr gly pro glu phe val thr gly tyr asn ile ile asn phe asp trp pro phe leu leu ala lys leu thr asp ile tyr lys val pro leu asp gly tyr gly arg met asn gly arg gly val phe arg val trp asp ile gly gln ser his phe gln lys arg tyr cys ile gln asp ser leu leu val gly gln leu phe phe lys phe leu pro his leu glu leu ser ala val ala arg leu ala gly ile asn leu leu arg leu ala asp gln lys gly phe ile leu pro asp thr gln thr ala gly arg his val gly tyr gln gly ala arg val leu asp pro leu tyr pro ser ile ile gln ala his asn leu cys phe ser thr leu ser leu arg ala asp ala val ala his leu glu ala gly lys asp tyr arg ile ile tyr gly asp thr asp ser ile phe val leu cys arg gly asn asn cys ala phe ile asn arg thr ser arg ala leu val asp leu ala phe gly ala val leu val asp ala his arg arg ile thr asp pro glu arg asp ile gln asp phe val leu thr ala glu leu ser val tyr tyr lys leu met ala arg arg ala gln val pro ser ile lys asp arg ile pro tyr val ile val ala gln thr arg glu val glu asp pro ala tyr ala ile ala his gly val ala leu asn thr asp tyr tyr phe ser his leu leu gly ala ala cys val thr phe lys ala leu phe gly asn asn ala lys ile thr glu ser leu leu lys arg phe ile pro glu val trp his pro pro asp asp val ala