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Humanparainfluenza virus type 1 is the major cause of croup ininfants and young children. There is currently neither vaccine norclinically effective treatment for parainfluenza virus infection.Hemagglutinin–neuraminidase glycoprotein is a key protein inviral infection, and its inhibition has been a target for 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en)-basedinhibitor development. In this study, we explore the effect of C-5modifications on the potency of Neu5Ac2en derivatives that targetthe human parainfluenza type-1 hemagglutinin–neuraminidaseprotein. Our study demonstrates that the replacement of the Neu5Ac2enC-5 acetamido moiety with more hydrophobic alkane-based moieties improvesthe inhibitory potency for both hemagglutinin–neuraminidasefunctions. These findings shed light on the importance of C-5 substitutionon Neu5Ac2en in the design of novel sialic acid-based inhibitors thattarget human parainfluenza type-1 hemagglutinin–neuraminidase. [ABSTRACT FROM AUTHOR]

HTS screening identified compound 2a(piperazinonederivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistancemapping studies suggested that this piperazinone chemotype targetsthe HCV nonstructural protein NS4B. Extensive SAR studies were performedaround 2aand the amide function and the C-3/C-6 cis stereochemistry of the piperazinone corewere essential for HCV activity. A 10-fold increase in GT-1 potencywas observed when the chiral phenylcyclopropyl amide side chain of 2awas replaced with p-fluorophenylisoxazole-carbonylmoiety (67). Replacing the C-6 nonpolarhydrophobic moiety of 67with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophenemoiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvementin GT-1b and 1a potency. However, the piperazonone class of compoundslacks GT-2 activity and, consequently, were not pursued further intodevelopment. [ABSTRACT FROM AUTHOR]

Toaid the design of next generation hepatitis C virus (HCV) drugs,the kinetics of the interactions between NS3 protease inhibitors andenzyme from genotypes 1a, 1b, and 3a have been characterized. Thelinear mechanism-based inhibitors VX-950 (telaprevir) and SCH 503034(boceprevir) benefited from covalent adduct formation. However, theapparent affinities were rather weak (VX-950, KD* of 340, 8.5, and 1000 nM for genotypes 1a, 1b and 3a, respectively;SCH 503034, KD* of 90 and 3.9 nM for 1band 3a, respectively). The non-mechanism-based macrocyclic inhibitorsBILN-2016 (ciluprevir) and ITMN-191 (danoprevir) had faster associationand slower dissociation kinetics, indicating that rigidification iskinetically favorable. ITMN-191 had nanomolar affinities for all genotypes(KD* of 0.13, 1.6, and 0.52 nM), suggestingthat a broad spectrum drug is conceivable. The data show that macrocyclicscaffolds and mechanism-based inhibition are advantageous but thatthere is considerable room for improvement of the kinetics of HCVprotease targeted drugs. [ABSTRACT FROM AUTHOR]

Described herein are structure–activityrelationship studiesthat resulted in the optimization of the activity of members of aclass of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors.Subsequent iterations of analogue design and syntheses successfullyaddressed off-target activities, most notably human pregnane X receptor(hPXR) transactivation, and led to significant improvements in thephysicochemical properties of lead compounds. Those analogues exhibitingimproved solubility and membrane permeability were shown to have notablyenhanced pharmacokinetic profiles. Additionally, a series of alkylbridged piperazine carboxamides was identified as being of particularinterest, and from which the compound BMS-791325 (2)was found to have distinguishing antiviral, safety, and pharmacokineticproperties that resulted in its selection for clinical evaluation. [ABSTRACT FROM AUTHOR]

Futuretreatments for individuals infected by the hepatitis C virus(HCV) will likely involve combinations of compounds that inhibit multipleviral targets. The helicase of HCV is an attractive target with noknown drug candidates in clinical trials. Herein we describe an integratedstrategy for identifying fragment inhibitors using structural andbiophysical techniques. Based on an X-ray structure of apo HCV helicaseand in silicoand bioinformatic analyses of HCV variants,we identified that one site in particular (labeled 3 + 4) was themost conserved and attractive pocket to target for a drug discoverycampaign. Compounds from multiple sources were screened to identifyinhibitors or binders to this site, and enzymatic and biophysicalassays (NMR and SPR) were used to triage the most promising ligandsfor 3D structure determination by X-ray crystallography. Medicinalchemistry and biophysical evaluations focused on exploring the mostpromising lead series. The strategies employed here can have generalutility in drug discovery. [ABSTRACT FROM AUTHOR]

By reducing the basicity of the coreheterocycle in a series ofHCV NS5B inhibitors, the hERG liability was reduced. The SAR was thensystematically explored in order to increase solubility and enabledose escalation while retaining potency. During this exploration,a facile decarboxylation was noted and was exploited as a novel prodrugmechanism. The synthesis and characterization of these prodrugs andtheir utilization in chronic toxicity studies are presented. [ABSTRACT FROM AUTHOR]

HepatitisC virus is a blood-borne infection and the leading causeof chronic liver disease (including cirrhosis and cancer) and livertransplantation. Since the identification of HCV in 1989, there hasbeen an extensive effort to identify and improve treatment options.An important milestone was reached in 2011 with the approval of thefirst-generation HCV NS3/4A protease inhibitors. However, new therapiesare needed to improve cure rates, shorten treatment duration, andimprove tolerability. Here we summarize the extensive medicinal chemistryeffort to develop novel P2 cyclopentane macrocyclic inhibitors guidedby HCV NS3 protease assays, the cellular replicon system, structure-baseddesign, and a panel of DMPK assays. The selection of compound 29(simeprevir, TMC435) as clinical candidate was based onits excellent biological, PK, and safety pharmacology profile. Compound 29has recently been approved for treatment of chronic HCVinfection in combination with pegylated interferon-α and ribavirinin Japan, Canada, and USA. [ABSTRACT FROM AUTHOR]

We have previously reported thatthe 6-aminoquinolone chemotypeis a privileged scaffold to obtain antibacterial and antiviral agents.Herein we describe the design, synthesis, and enzymatic and cellularcharacterization of new 6-aminoquinolone derivatives as potent inhibitorsof NS5B polymerase, an attractive and viable therapeutic target todevelop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolonederivative 8proved to be the best compound of this series,exhibiting an IC50value of 0.069 μM against NS5Bpolymerase and selective antiviral effect (EC50= 3.03μM) coupled with the absence of any cytostatic effect (CC50> 163 μM; SI > 54) in Huh 9–13 cellscarryinga HCV genotype 1b, as measured by MTS assay. These results indicatethat the 6-aminoquinolone scaffold is worthy of further investigationin the context of NS5B-targeted HCV drug discovery programs. [ABSTRACT FROM AUTHOR]

Herein we describe the design, synthesis,inhibitory potency, andpharmacokinetic properties of a novel class of achiral peptidomimeticHCV NS3 protease inhibitors. The compounds are based on a dipeptidomimeticpyrazinone glycine P3P2 building block in combination with an aromaticacyl sulfonamide in the P1P1′ position. Structure–activityrelationship data and molecular modeling support occupancy of theS2 pocket from elongated R6substituents on the 2(1H)-pyrazinone core and several inhibitors with improvedinhibitory potency down to Ki= 0.11 μMwere identified. A major goal with the design was to produce inhibitorsstructurally dissimilar to the di- and tripeptide-based HCV proteaseinhibitors in advanced stages of development for which cross-resistancemight be an issue. Therefore, the retained and improved inhibitorypotency against the drug-resistant variants A156T, D168V, and R155Kfurther strengthen the potential of this class of inhibitors. A numberof the inhibitors were tested in in vitro preclinical profiling assaysto evaluate their apparent pharmacokinetic properties. The variousR6substituents were found to have a major influence onsolubility, metabolic stability, and cell permeability. [ABSTRACT FROM AUTHOR]

HCV serine protease NS3 representsan attractive drug target because it is not only essential for viralreplication but also implicated in the viral evasion of the host immuneresponse pathway through direct cleavage of key proteins in the humaninnate immune system. Through structure-based drug design and optimization,macrocyclic peptidomimetic molecules bearing both a lipophilic P2isoindoline carbamate and a P1/P1′ acylsulfonamide/acylsulfamidecarboxylic acid bioisostere were prepared that possessed subnanomolarpotency against the NS3 protease in a subgenomic replicon-based cellularassay (Huh-7). Danoprevir (compound 49) was selectedas the clinical development candidate for its favorable potency profileacross multiple HCV genotypes and key mutant strains and for its goodin vitro ADME profiles and in vivo target tissue (liver) exposuresacross multiple animal species. X-ray crystallographic studies elucidatedseveral key features in the binding of danoprevir to HCV NS3 proteaseand proved invaluable to our iterative structure-based design strategy. [ABSTRACT FROM AUTHOR]

A reagent panel containing ten 4-substituted 4-nitrophenyl α-D-sialosides and a second panel of the corresponding sialic acid glycals were synthesized and used to probe the inhibition mechanism for two neuraminidases, the N2 enzyme from influenza type A virus and the enzyme from Micromonospora viridifaciens. For the viral enzyme the logarithm of the inhibition constant (Ki) correlated with neither the logarithm of the catalytic efficiency (kcat/Km) nor catalytic proficiency (kcat/Kmkun). These linear free energy relationship data support the notion that these inhibitors, which include the therapeutic agent Relenza, are not transition state mimics for the enzyme-catalyzed hydrolysis reaction. Moreover, for the influenza enzyme, a correlation (slope, 0.80 ± 0.08) is observed between the logarithms of the inhibition (Ki) and Michaelis (Km) constants. We conclude that the free energy for Relenza binding to the influenza enzyme mimics the enzyme-substrate interactions at the Michaelis complex. Thus, an influenza mutational response to a 4-substituted sialic acid glycal inhibitor can weaken the interactions between the inhibitor and the viral neuraminidase without a concomitant decrease in free energy of binding for the substrate at the enzyme-catalyzed hydrolysis transition state. The current findings make it clear that new structural motifs and/or substitution patterns need to be developed in the search for a bona fide influenza viral neuraminidase transition state analogue inhibitor. [ABSTRACT FROM AUTHOR]

The development of a practical, asymmetric synthesis of the hepatitis C virus (HCV) protease inhibitor MK-5172 (1), an 18-membered macrocycle, is described. [ABSTRACT FROM AUTHOR]

We have designed and synthesized new molecular tongsbased on arigid naphthalene scaffold and evaluated their antidimer activityon HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide(azatide) fragments into their peptidomimetic arms to reduce hydrophobicityand increase metabolic stability. These fragments are designed todisrupt the protein–protein interactions by reproducing thehydrogen bond pattern found in the antiparallel β-sheet formedbetween the N- and C-ends of thetwo monomers in the native PR. Kinetic analyses and fluorescent probebinding studies showed that several molecular tongs can inhibit PRdimerization. The best nonpeptidic molecular tongs to date were obtainedwith an inhibition constant Kidof 50nM for PR and 80 nM for the multimutated protease ANAM-11. The PRinhibition was selective, the aspartic proteases renin and pepsinwere not inhibited. [ABSTRACT FROM AUTHOR]

Nonnucleoside reverse transcriptase inhibitors (NNRTIs)are keycomponents of highly active antiretroviral therapy for the treatmentof HIV-1. A common problem with the first generation NNRTIs is theemergence of mutations in the HIV-1 reverse transcriptase (RT), inparticular, K103N and Y181C, which lead to resistance to the entireclass of inhibitor. Here we have evaluated the relative affinity ofthe newly designed NNRTI lersivirine (LRV) against drug-resistantmutations in HIV-1 RT using the molecular mechanics generalized Bornsurface area (MM-GBSA) method. Eight single and one double mutantvariants of RT are considered. Our results are in good agreement withexperimental results and yield insights into the mechanisms underlyingmutation-induced changes in the potency of LRV against RT. The strongest(54-fold) increase in the dissociation constant is found for the mutantF227C, originating from reduced electrostatic and van der Waals interactionsbetween LRV and RT as well as a higher energetic penalty from thedesolvation of polar groups. For the mutants K103N and Y181C onlya moderate (2-fold) increase in the dissociation constant is found,due to a balance of opposite changes in the polar solvation as wellas the electrostatic and van der Waals interactions between LRV andRT. The dissociation constant is decreased for the Y188C and G190A(2-fold), the M184V (5-fold), and the Y188C/Y188C mutant (10-fold),due to stronger electrostatic interactions between LRV and RT. Ourresults thus suggest that LRV is a highly potent and selective NNRTI,with excellent efficacy against NNRTI-resistant viruses, which isin agreement with experimental observations. [ABSTRACT FROM AUTHOR]

Neuroaminidase (NA) enzyme is a kind of glycoprotein that is found on the influenza A virus. During infection, NA is important for the release of influenza virions from the host cell surface together with viral aggregates. It may also be involved in targeting the virus to respiratory epithelial cells. In this study, a model electrochemical influenza A viral biosensor in which receptor-binding properties have been based on NA was developed for the first time. The biosensor's working principle is based on monitoring the interactions between fetuin A and NA enzyme. The assay was monitored step by step by using electrochemical impedance spectroscopy. [ABSTRACT FROM AUTHOR]

Fragment-based docking was used to select a conformation for virtual screening from a molecular dynamics trajectory of the West Nile virus nonstructural 3 protease. This conformation was chosen from an ensemble of 100 molecular dynamics snapshots because it optimally accommodates benzene, the most common ring in known drugs, and two positively charged fragments (methylguanidinium and 2-phenylimidazoline). The latter fragments were used as probes because of the large number of hydrogen bond acceptors in the substrate binding site of the protease. Upon high-throughput docking of a diversity set of 18 694 molecules and pose filtering, only five compounds were chosen for experimental validation, and two of them are active in the low micromolar range in an enzymatic assay and a tryptophan fluorescence quenching assay. Evidence for specific binding to the protease active site is provided by nuclear magnetic resonance spectroscopy. The two inhibitors have different scaffolds (diphenylurea and diphenyl ester) and are promising lead candidates because they have a molecular weight of about 300 Da. [ABSTRACT FROM AUTHOR]

HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c(KD= 0.79 nM), which has ∼20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c. [ABSTRACT FROM AUTHOR]

Knowing the substrate specificity of a protease is useful in determining its physiological substrates, developing robust assays, and designing specific inhibitors against the enzyme. In this work, we report the development of a combinatorial peptide library method for systematically profiling the substrate specificity of endopeptidases. A fluorescent donor (Edans) and quencher (Dabcyl) pair was added to the C- and N-termini of a support-bound peptide. Protease cleavage of the peptide removed the N-terminal quencher, resulting in fluorescent beads, which were isolated and individually sequenced by partial Edman degradation and mass spectrometry (PED-MS) to reveal the peptide sequence, as well as the site of proteolytic cleavage. The method was validated with bovine trypsin and Escherichia coli leader peptidase and subsequently applied to determine the substrate specificity of a viral protease, VP4, derived from the blotched snakehead virus (BSNV). The results show that VP4 cleaves peptides with a consensus sequence of (Abu/Ala/Pro)-X-Ala↓X, in agreement with the previously observed cleavage sites in its protein substrates. Resynthesis and a solution- phase assay of several representative sequences against VP4 confirmed the library screening results. [ABSTRACT FROM AUTHOR]

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F= 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30is a potent inhibitor in replicon assays, with EC50values of 10 and 6 nM against genotypes 1a and 1b, respectively. [ABSTRACT FROM AUTHOR]

We report the synthesis and structure−activity relationship (SAR) of a large series of acridones and acridone-fragment derivatives designed on the basis of the selective antihepatitis C virus (HCV) activity shown by acridone 2, previously studied as a potential antibovine viral diarrhea virus (BVDV) compound. The evaluation of their ability to inhibit the HCV replication in Huh-5-2 cells led to the identification of new, selective inhibitors. This indicates that the acridone skeleton, when properly functionalized, is a suitable scaffold to obtain potential anti-HCV agents. Interestingly, during identification of possible cellular and viral targets, it was discovered that compound 23exerts inhibitory activity on the HCV NS3 helicase, a very promising target for the development of anti-HCV drugs. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 μg/mL. [ABSTRACT FROM AUTHOR]

We report herein the synthesis of a series of 19 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives variously substituted at position 7 aimed at inhibiting selectively two-metal ion catalytic active sites. The compounds were tested against HIV-1 reverse transcriptase (RT) polymerase, HIV-1 RT ribonuclease H (RNase H), and HIV-1 integrase (IN). Most compounds displayed poor inhibition of RT polymerase even at 50 μM. The majority of the synthesized compounds inhibited RNase H and IN at micromolar concentrations, and some of them were weakly selective for IN. Surprisingly, two new hits were discovered, which displayed a high selectivity for IN with submicromolar IC50values. These enzymatic inhibitory properties may be related to the metal binding abilities of the compounds. Physicochemical studies were consistent with a 1/1 stoichiometry of the magnesium complexes in solution, and the metal complexation was strictly dependent on the enolization abilities of the compounds. Unfortunately, all tested compounds exhibited high cellular cytotoxicity in cell culture which limits their applications as antiviral agents. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Weak or nonexistent smallpox immunity in today's human population raises concerns about the possibility of natural or provoked genetic modifications leading to re-emergence of variola virus and other poxviruses. Thus, the development of new antiviral strategies aimed at poxvirus infections in humans is a high priority. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG is a rational therapeutic strategy for infections with poxviruses. Monkeypox virus, which occurs naturally in Africa, can cause a smallpox- like disease in humans. Here, the monkeypox virus UNG (mpUNG) is characterized and compared to vaccinia virus UNG (vUNG) and human UNG (hUNG). The mpUNG protein excises uracil preferentially from single-stranded DNA. Furthermore, mpUNG prefers the UG pair over the UA pair and does not excise oxidized bases. Both mpUNG and vUNG viral proteins are strongly inhibited by physiological concentrations of NaCI and MgCl2. Although the two viral DNA repair enzymes have similar substrate specificities, the kcat/KM values of mpUNG are higher than those of vUNG. The mpUNG protein was strongly inhibited by 5-azauracil and to a lesser extent by 4(6)-aminouracil and 5-halogenated uracil analogues, whereas uracil had no effect. To develop antiviral drugs toward mpUNG, we also validated a repair assay using the molecular beacons containing multiple uracil residues. Potential targets and strategies for combating pathogenic orthopoxviruses, including smallpox, are discussed. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-1-(dimethylamino)-1-methylethyl-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC95of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent. [ABSTRACT FROM AUTHOR]

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-β-D-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6- difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethyl- benzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed. [ABSTRACT FROM AUTHOR]

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn[sup 2+], the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG[sup º] ≈ -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼ 1.5 orders of magnitude (ΔG[sup º] ≈ -2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k[sub cat] term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides. [ABSTRACT FROM AUTHOR]

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein. [ABSTRACT FROM AUTHOR]

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration. [ABSTRACT FROM AUTHOR]

Examines the site specific DNA cleavage by Chlorella virus topoisomerase II. Frequency of the viral enzyme DNA cleavage; Use of PBCV-1 topoisomerase II; Display of PBCV-1 topoisomerase II sensitivity to anticancer drugs and DNA damage.

Describes the stereoselective routes of diastereomeric inhibitors of HIV protease, a monofluorinated analogues of the Merck HIV protease inhibitor indinavir. Stereoselective construction of the fluorinated core subunits by asymmetric alkylation reactions; Pharmacological properties of organic molecules; Inhibitory potency and biophysical properties of the protease inhibitors.

Reports on the kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase and its Y101F-mutant acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT). Demonstration of MCT as a lead compound in the design of resistance-repellent drugs for antiviral therapy.

Investigates the relationship between Mg2+-dependent activity and the self-assembly state of HIV-1 integrase using different protein preparations. Purification of the third preparation without detergent; Relationship of the ability of integrase to use Mg2+ as a cofactor with its self-assembly state in solution.

Investigates self-assembly properties of HIV-1 integrase by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. Accurate determination of long rotational correlation times with suitable photon counting; Oligomeric states of the widely used detergent-solubilized integrase in solution.

Determines whether aspartates 25 and 125, the active residues of HIV-1 protease, play a structural role that influences substrate processing. Production of a series of active site protease mutants in a cell-free protein synthesizing system; Proximity of the active site aspartate residues to the S1/S1' binding pocket.

Investigates the enzymatic activity of a C-terminally truncated form of the RNA-dependent RNA polymerase, termed NS5B(delta21), of the hepatitis C virus. Use of homopolymeric and heteropolymeric RNA templates; Comparability of the turnover number to those observed for other polymerases; Explanation for the discrepancy of the low, observed specific activity.

Reports on the quest for small nucleotide systems with nuclease stability of the intermediate phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase. Discovery of novel, nuclease-resistant dinucleotides with defined base sequences; Ability to inhibit the viral enzyme.

Investigates the mechanism-based inactivation of human liver cytochrome P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor. Identification of an active site residue of human liver P450 3A4; Indication that the residue of P450 3A4 adducted by L-754,394 is Glu307.

Examines the role of glutamine-125 (Gln-125) in herpes simplex virus type 1 thymidine kinase activity through the introduction of site-specific mutations of this residue to an aspartic acid, an asparagine or a glutamic acid. Reduction of thymidylate kinase activity in all three mutants.

Studies the potency of the slow binding enzyme inhibitors hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids in inhibiting the hepatitis C virus NS3 protease. Mechanism of inhibition; Determinant for the formation of collision complex; Selective participation of Lys 136 in stabilizing tight complex; Isomerization rate constant values of the collision complex.

Investigates whether interaction with the HIV accessory protein Nef induces activation of Src family kinases (Lyn, Fyn, Src and Lck) following coexpression with Nef in Rat-2 fibroblasts. Modulation of fibroblast transformation by coexpression of different Src family kinase members with Nef; Differential regulation of Src family kinase activity by Nef.

Presents evidence correlating the mobility of an HIV-1 integrase active site loop to catalytic activity. Covalent integration of the viral cDNA into the host chromosomal DNA in the replication of HIV-1; Conformational flexibility of the active site loop; Catalysis after DNA binding.

Examines whether covalently bound complex can be formed between the enzyme and the sialosyl cation intermediate in viral neuraminidase reaction via theoretical simulations. Applications for inhibitor designs; Hybrid potential free energy; Use of hybrid semiempirical quantum mechanical/molecular mechanical potential; Direct hydroxylation of the sialosyl cation.

Determines the solution structure of the DNA binding domain of HIV-1 integrase. Protein as a dimer in solution; Five-stranded beta-barrel with a topology similar to SH3 domain; Stacked beta interface.

Demonstrates that specific low efficiency UV photolinkage of GTP to the DIR 498-844 domain can be achieved. Theoretical background of experiment; Results of experiment; Discussion of results; References.

Presents the crystal structure of B/Lee/40 influenza virus neuraminidase (NA) and its complexes. Presumed N-acetylneuraminic acid (NANA) and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) at 1.8 Angstroms resolution; Binding of DANA and NANA to NA; Identity of moeity in active site.

Estimates contributions that influenza virus N9 neuraminidase residues make to complex formation. Comparison of calculated values to experimentally measured differences in monoclonal antibody binding between wild-type and mutated neuraminidases; Clustering of neuraminidase side chains around protruding surface regions.

Presents the determination of the rate of monomer interchange in a ligand-bound homodimeric protein from NOESY cross peaks. Identification of the two types of cross-peaks; Application to the HIV protease/KNI-529 complex, a viral enzyme; 2-fold symmetric inhibitors and asymmetric inhibitors; Crystal structures of the protease bound to symmetric inhibitors.