Impact of Integrase Sequences from HIV-1 Subtypes A6/A1 on the In Vitro Potency of Cabotegravir or Rilpivirine.

The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine versus daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).

GSK3732394: a Multi-specific Inhibitor of HIV Entry.

Long-acting antiretrovirals could provide a useful alternative to daily oral therapy for HIV-1-infected individuals. Building on a bi-specific molecule with adnectins targeting CD4 and gp41, a potential long-acting biologic, GSK3732394, was developed with three independent and synergistic modes of HIV entry inhibition that potentially could be self-administered as a long-acting subcutaneous injection. Starting with the bi-specific inhibitor, an α-helical peptide inhibitor was optimized as a linked molecule to the anti-gp41 adnectin, with each separate inhibitor exhibiting at least single-digit nanomolar (or lower) potency and a broad spectrum. Combination of the two adnectins and peptide activities into a single molecule was shown to have synergistic advantages in potency, the resistance barrier, and the ability to inhibit HIV-1 infections at low levels of CD4 receptor occupancy, showing that GSK3732394 can work in trans on a CD4+ T cell. Addition of a human serum albumin molecule prolongs the half-life in a human CD4 transgenic mouse, suggesting that it may have potential as a long-acting agent. GSK3732394 was shown to be highly effective in a humanized mouse model of infection. GSK3732394 is currently in clinical trials.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. Building on a bi-specific inhibitor approach targeting CD4 and gp41, a tri-specific molecule was generated with three distinct antiviral activities. The linkage of these three biologic inhibitors creates synergy that offers a series of advantages to the molecule. The addition of human serum albumin to the tri-specific inhibitor could allow it to function as a long-acting self-administered treatment for patients with HIV infection. This molecule is currently in early clinical trials.

A Novel gp41-Binding Adnectin with Potent Anti-HIV Activity Is Highly Synergistic when Linked to a CD4-Binding Adnectin.

The N17 region of gp41 in HIV-1 is the most conserved region in gp160. mRNA selection technologies were used to identify an adnectin that binds to this region and inhibits gp41-induced membrane fusion. Additional selection conditions were used to optimize the adnectin to greater potency (5.4 ± 2.6 nM) against HIV-1 and improved binding affinity for an N17-containing helical trimer (0.8 ± 0.4 nM). Resistance to this adnectin mapped to a single Glu-to-Arg change within the N17 coding region. The optimized adnectin (6200_A08) exhibited high potency and broad-spectrum activity against 123 envelope proteins and multiple clinical virus isolates, although certain envelope proteins did exhibit reduced susceptibility to 6200_A08 alone. The reduced potency could not be correlated with sequence changes in the target region and was thought to be the result of faster kinetics of fusion mediated by these envelope proteins. Optimized linkage of 6200_A08 with a previously characterized adnectin targeting CD4 produced a highly synergistic molecule, with the potency of the tandem molecule measured at 37 ± 1 pM. In addition, these tandem molecules now exhibited few potency differences against the same panel of envelope proteins with reduced susceptibility to 6200_A08 alone, providing evidence that they did not have intrinsic resistance to 6200_A08 and that coupling 6200_A08 with the anti-CD4 adnectin may provide a higher effective on rate for gp41 target engagement.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. This study describes the development and properties of an adnectin molecule that targets the most conserved region of the gp41 protein and inhibits HIV-1 with good potency. Moreover, when fused to a similar adnectin targeted to the human CD4 protein, the receptor for HIV-1, significant synergies in potency and efficacy are observed. These inhibitors are part of an effort to develop a larger biologic molecule that functions as a long-acting self-administered regimen for patients with HIV-1 infection.

The design, synthesis and structure-activity relationships associated with C28 amine-based betulinic acid derivatives as inhibitors of HIV-1 maturation.

The design and synthesis of a series of C28 amine-based betulinic acid derivatives as HIV-1 maturation inhibitors is described. This series represents a continuation of efforts following on from previous studies of C-3 benzoic acid-substituted betulinic acid derivatives as HIV-1 maturation inhibitors (MIs) that were explored in the context of C-28 amide substituents. Compared to the C-28 amide series, the C-28 amine derivatives exhibited further improvements in HIV-1 inhibitory activity toward polymorphisms in the Gag polyprotein as well as improved activity in the presence of human serum. However, plasma exposure of basic amines following oral administration to rats was generally low, leading to a focus on moderating the basicity of the amine moiety distal from the triterpene core. The thiomorpholine dioxide (TMD) 20 emerged from this study as a compound with the optimal antiviral activity and an acceptable pharmacokinetic profile in the C-28 amine series. Compared to the C-28 amide 3, 20 offers a 2- to 4-fold improvement in potency towards the screening viruses, exhibits low shifts in the EC50 values toward the V370A and ΔV370 viruses in the presence of human serum or human serum albumin, and demonstrates improved potency towards the polymorphic T371A and V362I virus variants.

Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity.

A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.

C-3 benzoic acid derivatives of C-3 deoxybetulinic acid and deoxybetulin as HIV-1 maturation inhibitors.

A series of C-3 phenyl- and heterocycle-substituted derivatives of C-3 deoxybetulinic acid and C-3 deoxybetulin was designed and synthesized as HIV-1 maturation inhibitors (MIs) and evaluated for their antiviral activity and cytotoxicity in cell culture. A 4-subsituted benzoic acid moiety was identified as an advantageous replacement for the 3'3'-dimethylsuccinate moiety present in previously disclosed MIs that illuminates new aspects of the topography of the pharmacophore. The new analogs exhibit excellent in vitro antiviral activity against wild-type (wt) virus and a lower serum shift when compared with the prototypical HIV-1 MI bevirimat (1, BVM), the first MI to be evaluated in clinical studies. Compound 9a exhibits comparable cell culture potency toward wt virus as 1 (WT EC50=16 nM for 9a compared to 10nM for 1). However, the potency of 9a is less affected by the presence of human serum, while the compound displays a similar pharmacokinetic profile in rats to 1. Hence 9a, the 4-benzoic acid derivative of deoxybetulinic acid, represents a new starting point from which to explore the design of a 2nd generation MI.

Characterization of clinical envelopes with lack of sensitivity to the HIV-1 inhibitors temsavir and ibalizumab.

Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC50) and ibalizumab sensitivity by IC50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to ∼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab.

Activity of the HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068, against CD4-independent viruses and HIV-1 envelopes resistant to other entry inhibitors.

BMS-626529 is a novel small-molecule HIV-1 attachment inhibitor active against both CCR5- and CXCR4-tropic viruses. BMS-626529 functions by preventing gp120 from binding to CD4. A prodrug of this compound, BMS-663068, is currently in clinical development. As a theoretical resistance pathway to BMS-663068 could be the development of a CD4-independent phenotype, we examined the activity of BMS-626529 against CD4-independent viruses and investigated whether resistance to BMS-626529 could be associated with a CD4-independent phenotype. Finally, we evaluated whether cross-resistance exists between BMS-626529 and other HIV-1 entry inhibitors. Two laboratory-derived envelopes with a CD4-independent phenotype (one CXCR4 tropic and one CCR5 tropic), five envelopes from clinical isolates with preexisting BMS-626529 resistance, and several site-specific mutant BMS-626529-resistant envelopes were examined for their dependence on CD4 for infectivity or susceptibility to BMS-626529. Viruses resistant to other entry inhibitors (enfuvirtide, maraviroc, and ibalizumab) were also examined for susceptibility to BMS-626529. Both CD4-independent laboratory isolates retained sensitivity to BMS-626529 in CD4(-) cells, while HIV-1 envelopes from viruses resistant to BMS-626529 exhibited no evidence of a CD4-independent phenotype. BMS-626529 also exhibited inhibitory activity against ibalizumab- and enfuvirtide-resistant envelopes. While there appeared to be some association between maraviroc resistance and reduced susceptibility to BMS-626529, an absolute correlation cannot be presumed, since some CCR5-tropic maraviroc-resistant envelopes remained sensitive to BMS-626529. Clinical use of the prodrug BMS-663068 is unlikely to promote resistance via generation of CD4-independent virus. No cross-resistance between BMS-626529 and other HIV entry inhibitors was observed, which could allow for sequential or concurrent use with different classes of entry inhibitors.

In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

Evaluation of HIV-1 inhibition by stereoisomers and analogues of the sesquiterpenoid hydroquinone peyssonol A.

Peyssonol A, a brominated natural product with documented anti-HIV-1 activity, was synthesized racemically along with 6 isomers and 15 truncated analogues and synthetic precursors. These compounds were screened in a cell-based assay against a recombinant HIV-1 strain to investigate structure-activity relationships. The results obtained suggest that both the aliphatic and aromatic domains of peyssonol A are responsible for its potency, while the stereochemical configuration of the substituents on the aliphatic domain, including their bromine atom, are largely irrelevant. Although none of the analogues tested were as potent as the parent natural product, several exhibited greater therapeutic indices due to reduced cytotoxicity, noting that nearly all compounds tested were measurably cytotoxic.

Clinical evidence for a lack of cross-resistance between temsavir and ibalizumab or maraviroc.

BACKGROUND: Temsavir (TMR), the active agent of the gp120-directed attachment inhibitor fostemsavir (FTR), the CD4-directed attachment inhibitor ibalizumab (IBA), and the CCR5 antagonist maraviroc (MVC) are antiretroviral agents that target steps in HIV-1 viral entry. Although mechanisms of inhibition of the three agents are different, it is important to understand whether there is potential for cross-resistance between these agents, as all involve interactions with gp120. METHODS: Envelopes derived from plasma samples from participants in the BRIGHTE study who experienced protocol-derived virologic failure (PDVF) and were co-dosed with FTR and either IBA or MVC were analyzed for susceptibility to the agents. Also, CCR5-tropic MVC-resistant envelopes from the MOTIVATE trials were regenerated and studies were performed to understand whether susceptibility to multiple agents were linked. RESULTS: The cloned envelopes exhibited reduced susceptibility to TMR and resistance to the co-dosed agent. At PDVF, emergent or preexisting amino acid substitutions were present at TMR positions of interest. When amino acid substitutions at these positions were reverted to the consensus sequence, full susceptibility to TMR was restored without effecting resistance to the co-dosed agent. In addition, five envelopes from MOTIVATE were regenerated and exhibited R5-tropic-MVC-resistance. Only one exhibited reduced susceptibility to TMR and it contained an M426L polymorphism. When reverted to 426M, full sensitivity for TMR was restored, but it remained MVC resistant. CONCLUSION: The data confirm that decreased susceptibility to TMR and resistance to IBA or MVC are not linked and that there is no cross-resistance between either of these two agents and FTR.

Synthesis, Structure-Activity Relationships, and In Vivo Evaluation of Novel C-17 Amine Derivatives Based on GSK3640254 as HIV-1 Maturation Inhibitors with Broad Spectrum Activity.

An investigation of the structure-activity relationships of a series of HIV-1 maturation inhibitors (MIs) based on GSK3640254 (4) was conducted by incorporating novel C-17 amine substituents to reduce the overall basicity of the resultant analogues. We found that replacement of the distal amine on the C-17 sidechain present in 4 with a tertiary alcohol in combination with either a heterocyclic ring system or a cyclohexyl ring substituted with polar groups provided potent wild-type HIV-1 MIs that also retained excellent potency against a T332S/V362I/prR41G variant, a laboratory strain that served as a surrogate to assess HIV-1 polymorphic virus coverage. Compound 26 exhibited broad-spectrum HIV-1 activity against an expanded panel of clinically relevant Gag polymorphic viruses and had the most desirable overall profile in this series of compounds. In pharmacokinetic studies, 26 had low clearance and exhibited 24 and 31% oral bioavailability in rats and dogs, respectively.

The Discovery of GSK3640254, a Next-Generation Inhibitor of HIV-1 Maturation.

GSK3640254 is an HIV-1 maturation inhibitor (MI) that exhibits significantly improved antiviral activity toward a range of clinically relevant polymorphic variants with reduced sensitivity toward the second-generation MI GSK3532795 (BMS-955176). The key structural difference between GSK3640254 and its predecessor is the replacement of the para-substituted benzoic acid moiety attached at the C-3 position of the triterpenoid core with a cyclohex-3-ene-1-carboxylic acid substituted with a CH2F moiety at the carbon atom α- to the pharmacophoric carboxylic acid. This structural element provided a new vector with which to explore structure-activity relationships (SARs) and led to compounds with improved polymorphic coverage while preserving pharmacokinetic (PK) properties. The approach to the design of GSK3640254, the development of a synthetic route and its preclinical profile are discussed. GSK3640254 is currently in phase IIb clinical trials after demonstrating a dose-related reduction in HIV-1 viral load over 7-10 days of dosing to HIV-1-infected subjects.

Design, Synthesis, and SAR of C-3 Benzoic Acid, C-17 Triterpenoid Derivatives. Identification of the HIV-1 Maturation Inhibitor 4-((1 R,3a S,5a R,5b R,7a R,11a S,11b R,13a R,13b R)-3a-((2-(1,1-Dioxidothiomorpholino)ethyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1 H-cyclopenta[ a]chrysen-9-yl)benzoic Acid (GSK3532795, BMS-955176).

GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC50 <15 nM toward a panel of common polymorphisms representative of 96.5% HIV-1 subtype B virus) with a favorable pharmacokinetic profile in preclinical species.

Discovery of BMS-955176, a Second Generation HIV-1 Maturation Inhibitor with Broad Spectrum Antiviral Activity.

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.

Pyrazolo-Piperidines Exhibit Dual Inhibition of CCR5/CXCR4 HIV Entry and Reverse Transcriptase.

We report novel anti-HIV-1 agents with combined dual host-pathogen pharmacology. Lead compound 3, composed of a pyrazole-piperidine core, exhibits three concurrent mechanisms of action: (1) non-nucleoside reverse transcriptase inhibition, (2) CCR5-mediated M-tropic viral entry inhibition, and (3) CXCR4-based T-tropic viral entry inhibition that maintains native chemokine ligand binding. This discovery identifies important tool compounds for studying viral infectivity and prototype agents that block HIV-1 entry through dual chemokine receptor ligation.

Biochemical analysis of HIV-1 integrase variants resistant to strand transfer inhibitors.

In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k(p)/K(m)) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k(p)) values. However, smaller second-order effects were caused by up to 4-fold increases in K(m) values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3'-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors.

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Respiratory syncytial virus fusion inhibitors. Part 5: Optimization of benzimidazole substitution patterns towards derivatives with improved activity.

Extensive SAR studies and optimization of ADME properties of benzimidazol-2-one derivatives led to the identification of BMS-433771 (3) as an orally active RSV fusion inhibitor. In order to extend the structure-activity relationships for this compound series, substitution of the benzimidazole ring was examined with a view to establishing additional productive interactions between the inhibitor and functionality present in the proposed binding pocket. Amongst the compounds synthesized, the 5-aminomethyl analogue 10aa demonstrated potent antiviral activity towards wild-type RSV and retained excellent inhibitory activity towards a virus that had been developed to express resistance to BMS-433771 (3), data consistent with an additional productive interaction between the inhibitor and the fusion protein target.