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.

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.

Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.

HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.

A Probody T Cell-Engaging Bispecific Antibody Targeting EGFR and CD3 Inhibits Colon Cancer Growth with Limited Toxicity.

T cell-engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are "masked" to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications. SIGNIFICANCE: A conditionally active EGFR-CD3 T cell-engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1.

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

Discovery of an Atropisomeric PI3Kß Selective Inhibitor through Optimization of the Hinge Binding Motif.

A series of PI3Kß selective inhibitors derived from a novel 4-(1H-benzo[d]imidazol-1-yl)quinoline chemotype has been rationally designed. Crucial to achieving the desired selectivity over the other class I PI3K isoforms, including the challenging δ-isoform, was the identification of a subset of substituted pyridine hinge binders. This work led to the discovery of (P)-14, a highly selective and orally bioavailable PI3Kß inhibitor displaying an excellent pharmacokinetic profile in addition to great cellular potency in various PTEN-deficient tumor cell lines. Results from a dog toxicology study revealing structure-related, off-target ocular toxicity are also briefly discussed.

Atropisomerism by Design: Discovery of a Selective and Stable Phosphoinositide 3-Kinase (PI3K) ß Inhibitor.

Atropisomerism is a type of axial chirality in which enantiomers or diastereoisomers arise due to hindered rotation around a bond axis. In this manuscript, we report a case in which torsional scan studies guided the thoughtful creation of a restricted axis of rotation between two heteroaromatic systems of a phosphoinositide 3-kinase (PI3K) ß inhibitor, generating a pair of atropisomeric compounds with significantly different pharmacological and pharmacokinetic profiles. Emblematic of these differences, the metabolism of inactive ( M)-28 is primarily due to the cytosolic enzyme aldehyde oxidase, while active ( P)-28 has lower affinity for aldehyde oxidase, resulting in substantially better metabolic stability. Additionally, we report torsional scan and experimental studies used to determine the barriers of rotation of this novel PI3Kß inhibitor.

Discovery of a Phosphoinositide 3-Kinase (PI3K) ß/δ Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kß Potency in a PI3Kδ-Selective Template by Targeting Nonconserved Asp856.

Phosphoinositide 3-kinase (PI3K) ß signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kß/δ inhibitors in which PI3Kß potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kß/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

Discovery of Orally Efficacious Phosphoinositide 3-Kinase δ Inhibitors with Improved Metabolic Stability.

Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

2,4,6-Triaminopyrimidine as a Novel Hinge Binder in a Series of PI3Kδ Selective Inhibitors.

Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.

Optimized high-throughput screen for hepatitis C virus translation inhibitors.

Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5' capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, as well we the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.

Screening and identification of a novel class of TGF-ß type 1 receptor kinase inhibitor.

Transforming growth factor ß (TGF-ß) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2ß]pyridazin 6-amine) with a low IC(50) value of 0.7 µM. Compound 8 also inhibited the TGF-ß-induced nuclear translocation of SMAD with an EC(50) value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.