Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells.

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. METHODS: Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. RESULTS: Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M-1 s-1 for BTK with selectivity against important off-targets. CONCLUSIONS: For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. GENERAL SIGNIFICANCE: This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Homogeneous BTK Occupancy Assay for Pharmacodynamic Assessment of Tirabrutinib (GS-4059/ONO-4059) Target Engagement.

Bruton's tyrosine kinase (BTK) is a clinically validated target for B-cell leukemias and lymphomas with FDA-approved small-molecule inhibitors ibrutinib and acalabrutinib. Tirabrutinib (GS-4059/ONO-4059, Gilead Sciences, Inc., Foster City, CA) is a second-generation, potent, selective, irreversible BTK inhibitor in clinical development for lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL). An accurate pharmacodynamic assay to assess tirabrutinib target coverage in phase 1/2 clinical studies will inform dose and schedule selection for advanced clinical evaluation. We developed a novel duplex homogeneous BTK occupancy assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to measure free and total BTK levels in a multiplexed format. The dual-wavelength emission property of terbium-conjugated anti-BTK antibody served as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra. The assay was characterized and qualified using full-length purified recombinant human BTK protein and peripheral blood mononuclear cells derived from healthy volunteers and patients with CLL. We demonstrated assay utility using cells derived from lymph node and bone marrow samples from patients with CLL and DLBCL. Our TR-FRET-based BTK occupancy assay provides accurate, quantitative assessment of BTK occupancy in the clinical trial program for tirabrutinib and is in use in ongoing clinical studies.

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A.

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.

Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase δ.

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.

Amide-based inhibitors of p38alpha MAP kinase. Part 2: design, synthesis and SAR of potent N-pyrimidyl amides.

Optimization of a tri-substituted N-pyridyl amide led to the discovery of a new class of potent N-pyrimidyl amide based p38alpha MAP kinase inhibitors. Initial SAR studies led to the identification of 5-dihydrofuran as an optimal hydrophobic group. Additional side chain modifications resulted in the introduction of hydrogen bond interactions. Through extensive SAR studies, analogs bearing free amino groups and alternatives to the parent (S)-alpha-methyl benzyl moiety were identified. These compounds exhibited improved cellular activities and maintained balance between p38alpha and CYP3A4 inhibition.

Design and synthesis of piperazine-indole p38 alpha MAP kinase inhibitors with improved pharmacokinetic profiles.

Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38alpha MAP kinase inhibitors are described. Biological evaluation of these compounds focused on maintaining activity while improving pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions on the benzylic methylene.

Piperidine-based heterocyclic oxalyl amides as potent p38 alpha MAP kinase inhibitors.

The design and synthesis of a new class of p38alpha MAP kinase inhibitors based on 4-fluorobenzylpiperidine heterocyclic oxalyl amides are described. Many of these compounds showed low-nanomolar activities in p38alpha enzymatic and cell-based cytokine TNFalpha production inhibition assays. The optimal linkers between the piperidine and the oxalyl amide were found to be [6,5] fused ring heterocycles. Substituted indoles and azaindoles were favored structural motifs in the cellular assay.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

Aryl-indolyl maleimides as inhibitors of CaMKIIdelta. Part 3: Importance of the indole orientation.

A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 10nM to >20microM and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core of the inhibitors. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed.

Pyrimidine-based inhibitors of CaMKIIdelta.

Non-ATP competitive pyrimidine-based inhibitors of CaMKIIdelta were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.

Aryl-indolyl maleimides as inhibitors of CaMKIIdelta. Part 1: SAR of the aryl region.

A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 34nM to >20microM and were dependant upon both the nature of the aryl group and the hydrogen bond donating potential of the maleimide ring. Key interactions with the kinase ATP site and hinge region were found to be consistent with homology modeling predictions.

Aryl-indolyl maleimides as inhibitors of CaMKIIdelta. Part 2: SAR of the amine tether.

A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin-dependant kinase. Inhibitory activities against the enzyme ranged from 34nM to >20microM and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed.

Triazole derivatives as non-nucleoside inhibitors of HIV-1 reverse transcriptase--structure-activity relationships and crystallographic analysis.

A series of 3,4,5-trisubstituted 1,2,4-4H triazole derivatives was synthesized and investigated for HIV-1 reverse transcriptase inhibition. An X-ray structure with HIV-1 RT secured the binding mode and allowed the key interactions with the enzyme to be identified.