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.).

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.

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.

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.

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.

Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors.

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.

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+).

Discovery of a potent boronic acid derived inhibitor of the HCV RNA-dependent RNA polymerase.

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.

Rational design of potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A new series of non-nucleoside reverse transcriptase inhibitors based on an imidazole-amide biarylether scaffold has been identified and shown to possess potent antiviral activity against HIV-1, including the NNRTI-resistant Y188L mutated virus. X-ray crystallography of inhibitors bound to reverse transcriptase, including a structure of the Y188L RT protein, was used extensively to help identify and optimize the key hydrogen-bonding motif. This led directly to the design of compound 43 that exhibits remarkable antiviral activity (EC50<1 nM) against a wide range of NNRTI-resistant viruses and a favorable pharmacokinetic profile across multiple species.

Structure guided design of 5-arylindazole glucocorticoid receptor agonists and antagonists.

Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

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.

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.

Kinase-targeted library design through the application of the PharmPrint methodology.

The PharmPrint methodology, as modified and implemented by Deanda and Stewart, was prospectively evaluated for use as a virtual high-throughput screening tool by applying it to the design of target-focused arrays. To this end, PharmPrint quantitative structure-activity relationship (QSAR) models for the prediction of AKT1, Aurora-A, and ROCK1 inhibition were constructed and used to virtually screen two large combinatorial libraries. Based on predicted activities, an Aurora-A targeted array and a ROCK1 targeted array were designed and synthesized. One control group per designed array was also synthesized to assess the enrichment levels achieved by the QSAR models. For the Aurora-A targeted array, the hit rate, against the intended target, was 42.9%, whereas that of the control group was 0%. Thus, the enrichment level achieved by the Aurora-A QSAR model was incalculable. For the ROCK1 targeted array, the hit rate against the intended target was 30.6%, whereas that of the control group was 5.10%, making the enrichment level achieved by the ROCK1 QSAR model 6-fold above control. Clearly, these results support the use of the PharmPrint methodology as a virtual screening tool for the design of kinase-targeted arrays.

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).

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.