Integrase Inhibitor Resistance Mechanisms and Structural Characteristics in Antiretroviral Therapy-Experienced, Integrase Inhibitor-Naive Adults with HIV-1 Infection Treated with Dolutegravir plus Two Nucleoside Reverse Transcriptase Inhibitors in the DAWNING Study.

At week 48 in the phase IIIb DAWNING study, the integrase strand transfer inhibitor (INSTI) dolutegravir plus 2 nucleoside reverse transcriptase inhibitors demonstrated superiority to ritonavir-boosted lopinavir in achieving virologic suppression in adults with HIV-1 who failed first-line therapy. Here, we report emergent HIV-1 drug resistance and mechanistic underpinnings among dolutegravir-treated adults in DAWNING. Population viral genotyping, phenotyping, and clonal analyses were performed on participants meeting confirmed virologic withdrawal (CVW) criteria on dolutegravir-containing regimens. Dolutegravir binding to and structural changes in HIV-1 integrase-DNA complexes with INSTI resistance-associated substitutions were evaluated. Of participants who received dolutegravir through week 48 plus an additional 110 weeks for this assessment, 6 met CVW criteria with treatment-emergent INSTI resistance-associated substitutions and 1 had R263R/K at baseline but not at CVW. All 7 achieved HIV-1 RNA levels of <400 copies/mL (5 achieved <50 copies/mL) before CVW. Treatment-emergent G118R was detected in 5 participants, occurring with ≥2 other integrase substitutions, including R263R/K, in 3 participants and without other integrase substitutions in 2 participants. G118R or R263K increased the rate of dolutegravir dissociation from integrase-DNA complexes versus wild-type but retained prolonged binding. Overall, among treatment-experienced adults who received dolutegravir in DAWNING, 6 of 314 participants developed treatment-emergent INSTI resistance-associated substitutions, with a change in in vitro dolutegravir resistance of >10-fold and reduced viral replication capacity versus baseline levels. This study demonstrates that the pathway to dolutegravir resistance is a challenging balance between HIV-1 phenotypic change and associated loss of viral fitness. (This study has been registered at ClinicalTrials.gov under identifier NCT02227238.).

Emergence of Resistance in HIV-1 Integrase with Dolutegravir Treatment in a Pediatric Population from the IMPAACT P1093 Study.

P1093 is a multicenter, open-label, phase I/II study of pharmacokinetics, safety, and tolerability of dolutegravir plus an optimized background regimen in pediatric participants aged 4 weeks to <18 years with HIV-1. Most participants were highly treatment experienced. We report the mechanisms of emergent integrase strand transfer inhibitor (INSTI) resistance among adolescents and children receiving dolutegravir. Plasma was collected at screening and near protocol-defined virologic failure (PDVF) for population-level and, for some samples, clonal-level integrase genotyping, phenotyping, and replication capacity. HIV-1 RNA was assessed in all available plasma samples. Phylogenetic analysis of clonal integrase sequences and homology modeling of HIV-1 intasome complexes containing resistance-associated substitutions were performed. Treatment-emergent INSTI resistance was detected in 8 participants who met PDVF criteria. The rare INSTI resistance-associated substitution G118R or R263K developed in 6 participants. The on-study secondary integrase substitution E157Q or L74I was observed in 2 participants. G118R reduced dolutegravir susceptibility and integrase replication capacity more than R263K and demonstrated greater reduction in susceptibility and integrase replication capacity when present with specific secondary integrase substitutions, including L74M, T66I, and E138E/K. Continuing evolution after R263K acquisition led to reduced dolutegravir susceptibility and integrase replication capacity. Structural examination revealed potential mechanisms for G118R- and R263K-mediated INSTI resistance. G118R and R263K INSTI resistance substitutions, which are distinct to second-generation INSTIs, were detected in adolescents and children with prior virologic failure who received dolutegravir. This study provides additional molecular and structural characterization of integrase to aid in the understanding of INSTI resistance mechanisms in antiretroviral-experienced populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01302847.).

Susceptibility of global HIV-1 clinical isolates to fostemsavir using the PhenoSense® Entry assay.

BACKGROUND: Fostemsavir is a prodrug of a first-in-class HIV-1 attachment inhibitor, temsavir, that binds to gp120 and blocks attachment to the host-cell CD4 receptor, preventing entry and infection of the target cell. Previous studies using a limited number of clinical isolates showed that there was intrinsic variability in their susceptibility to temsavir. OBJECTIVES: Here, an analysis was performed using all clinical isolates analysed in the Monogram Biosciences PhenoSense® Entry assay as part of the development programme. METHODS: In total, 1337 individual envelopes encompassing 20 different HIV-1 subtypes were examined for their susceptibility to temsavir. However, only seven subtypes (B, C, F1, A, [B, F1], BF and A1) were present more than five times, with subtype B (881 isolates) and subtype C (156 isolates) having the largest numbers. RESULTS: As expected, variability in susceptibility was observed within all subtypes. However, for the great majority of these viruses, temsavir was highly potent, with most viruses exhibiting IC50s <10 nM. One exception was CRF01_AE viruses, where all five isolates exhibited IC50s >100 nM. For the 607 isolates where tropism data were available, geometric mean temsavir IC50 values were remarkably similar for CCR5-, CXCR4- and dual mixed-tropic envelopes from infected individuals. CONCLUSIONS: These data show that HIV-1 viruses from most subtypes are highly susceptible to temsavir and that temsavir susceptibility is independent of tropism.

A knowledge-based, structural-aided discovery of a novel class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors.

Through an internal virtual screen at GlaxoSmithKline a distinct class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors were discovered. Careful evaluation of crystal structures and SAR led to a novel, potent, and orally active imidazopyridine inhibitor of H-PGDS, 20b. Herein, describes the identification of 2 classes of inhibitors, their syntheses, and their challenges.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

2,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships.

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10-100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.

A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157.

hCD157 catalyzes the hydrolysis of nicotinamide riboside (NR) and nicotinic acid riboside (NAR). The release of nicotinamide or nicotinic acid from NR or NAR was confirmed by spectrophotometric, HPLC and NMR analyses. hCD157 is inactivated by a mechanism-based inhibitor, 2'-deoxy-2'-fluoro-nicotinamide arabinoside (fNR). Modification of the enzyme during the catalytic cycle by NR, NAR, or fNR increased the intrinsic protein fluorescence by approximately 50%. Pre-steady state and steady state data were used to derive a minimal kinetic scheme for the hydrolysis of NR. After initial complex formation a reversible step (360 and 30s(-1)) is followed by a slow irreversible step (0.1s(-1)) that defined the rate limiting step, or kcat. The calculated KMapp value for NR in the hydrolytic reaction is 6nM. The values of the kinetic constants suggest that one biological function of cell-surface hCD157 is to bind and slowly hydrolyze NR, possibly converting it to a ligand-activated receptor. Differences in substrate preference between hCD157 and hCD38 were rationalized through a comparison of the crystal structures of the two proteins. This comparison identified several residues in hCD157 (F108 and F173) that can potentially hinder the binding of dinucleotide substrates (NAD+).

The sensitivity of HIV-1 gp120 polymorphs to inhibition by temsavir correlates to temsavir binding on-rate.

Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. Fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r = 0.7332; P = 0.0246) as well as temsavir binding on-rate (r = -0.8940; P = 0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.

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.

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment.

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4's flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

Improving the developability profile of pyrrolidine progesterone receptor partial agonists.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.

X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain.

An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17 alpha furoate ester to occupy more fully the lipophilic 17 alpha pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.

The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist.

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Novel secondary mutations C56S and G149A confer resistance to HIV-1 integrase strand transfer inhibitors.

Cabotegravir (CAB, S/GSK1265744) is an investigational second-generation integrase strand transfer inhibitor (INSTI) with a chemical structure similar to dolutegravir. CAB is under development as a long-acting injectable formulation for treatment of HIV-1 infection and for pre-exposure prophylaxis. We conducted an in vitro passage study of raltegravir- or elvitegravir-resistant signature mutants in the presence of CAB to characterize the resistance profile of this drug. During passage with Q148H virus, G140S arose by day 14, followed by G149A and C56S. Using site-directed mutagenesis, we obtained HIV molecular clones containing mutations encoding C56S and G149A in the integrase-coding region. Those substitutions were characterized in vitro as INSTI-resistance-associated secondary resistance mutations. Signature mutant viruses G140S/Q148H in which C56S and G149A were added acquired further INSTI resistance in conjunction with diminished integration activity, which yielded slower growth under drug-free conditions.

Discovery of 4-Amino-8-quinoline Carboxamides as Novel, Submicromolar Inhibitors of NAD-Hydrolyzing Enzyme CD38.

Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.