Discovery of 4,5,6,7-Tetrahydropyrazolo[1.5-a]pyrizine Derivatives as Core Protein Allosteric Modulators (CpAMs) for the Inhibition of Hepatitis B Virus.

Hepatitis B Virus (HBV) core protein allosteric modulators (CpAMs) are an attractive class of potential anti-HBV therapeutic agents. Here we describe the efforts toward the discovery of a series of 4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine (THPP) compounds as HBV CpAMs that effectively inhibit a broad range of nucleos(t)ide-resistant HBV variants. The lead compound 45 demonstrated inhibition of HBV DNA viral load in a HBV AAV mouse model by oral administration.

Discovery of Linvencorvir (RG7907), a Hepatitis B Virus Core Protein Allosteric Modulator, for the Treatment of Chronic HBV Infection.

Described herein is the first-time disclosure of Linvencorvir (RG7907), a clinical compound and a hepatitis B virus (HBV) core protein allosteric modulator, for the treatment of chronic HBV infection. Built upon the core structure of hetero aryl dihydropyrimidine, RG7907 was rationally designed by combining all the drug-like features of low CYP3A4 induction, potent anti-HBV activity, high metabolic stability, low hERG liability, and favorable animal pharmacokinetic (PK) profiles. In particular, the chemistry strategy to mitigate CYP3A4 induction through introducing a large, rigid, and polar substituent at the position that has less interaction with the therapeutic biological target (HBV core proteins herein) is of general interest to the medicinal chemistry community. RG7907 demonstrated favorable animal PK, pharmacodynamics, and safety profiles with sufficient safety margins supporting its clinical development in healthy volunteers and HBV-infected patients.

Translation Potential and Challenges of In Vitro and Murine Models in Cancer Clinic.

As one of the leading causes of death from disease, cancer continues to pose a serious threat to human health globally. Despite the development of novel therapeutic regimens and drugs, the long-term survival of cancer patients is still very low, especially for those whose diagnosis is not caught early enough. Meanwhile, our understanding of tumorigenesis is still limited. Suitable research models are essential tools for exploring cancer mechanisms and treatments. Herein we review and compare several widely used in vitro and in vivo murine cancer models, including syngeneic tumor models, genetically engineered mouse models (GEMM), cell line-derived xenografts (CDX), patient-derived xenografts (PDX), conditionally reprogrammed (CR) cells, organoids, and MiniPDX. We will summarize the methodology and feasibility of various models in terms of their advantages and limitations in the application prospects for drug discovery and development and precision medicine.

Discovery of Pyrido[2,3-b]indole Derivatives with Gram-Negative Activity Targeting Both DNA Gyrase and Topoisomerase IV.

The rise of multidrug resistant (MDR) Gram-negative (GN) pathogens and the decline of available antibiotics that can effectively treat these severe infections are a major threat to modern medicine. Developing novel antibiotics against MDR GN pathogens is particularly difficult as compounds have to permeate the GN double membrane, which has very different physicochemical properties, and have to circumvent a plethora of resistance mechanisms such as multiple efflux pumps and target modifications. The bacterial type II topoisomerases DNA gyrase (GyrA2B2) and Topoisomerase IV (ParC2E2) are highly conserved targets across all bacterial species and validated in the clinic by the fluoroquinolones. Dual inhibitors targeting the ATPase domains (GyrB/ParE) of type II topoisomerases can overcome target-based fluoroquinolone resistance. However, few ATPase inhibitors are active against GN pathogens. In this study, we demonstrated a successful strategy to convert a 2-carboxamide substituted azaindole chemical scaffold with only Gram-positive (GP) activity into a novel series with also potent activity against a range of MDR GN pathogens. By systematically fine-tuning the many physicochemical properties, we identified lead compounds such as 17r with a balanced profile showing potent GN activity, high aqueous solubility, and desirable PK features. Moreover, we showed the bactericidal efficacy of 17r using a neutropenic mouse thigh infection model.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Discovery of Ziresovir as a Potent, Selective, and Orally Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor.

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.

Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action.

Chronic hepatitis B virus (HBV) infection is a serious public health burden, and current therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel therapeutic agents with differentiated mechanism of action (MOA) from the current standard of care. RG7834, a compound from the dihydroquinolizinone (DHQ) chemical series, is a first-in-class highly selective and orally bioavailable HBV inhibitor which can reduce both viral antigens and viral DNA with a novel mechanism of action. Here we report the discovery of RG7834 from a phenotypic screening and the structure-activity relationship (SAR) of the DHQ chemical series. RG7834 can selectively inhibit HBV but not other DNA or RNA viruses in a virus panel screening. Both in vitro and in vivo profiles of RG7834 are described herein, and the data support further development of this compound as a chronic HBV therapy.

Discovery of Benzoazepinequinoline (BAQ) Derivatives as Novel, Potent, Orally Bioavailable Respiratory Syncytial Virus Fusion Inhibitors.

A novel benzoazepinequnoline (BAQ) series was discovered as RSV fusion inhibitors. BAQ series originated from compound 2, a hit from similarity-based virtual screening. In SAR exploration, benzoazepine allowed modifications in the head moiety. Benzylic sulfonyl on benzoazepine and 6-Me on quinoline were crucial for good anti-RSV activity. Although the basic amine in the head portion was crucial for anti-RSV activity, the attenuated basicity was required to reduce Vss. Introducing oxetane to the head portion led to discovery of compound 1, which demonstrated single-digit nM anti-RSV activity against different RSV strains, reasonable oral exposure in plasma, and 78-fold higher exposure in lung. Compound 1 also displayed 1 log viral reduction in a female BALB/c mice RSV model by b.i.d. oral dosing at 12.5 mg/kg. A single resistant mutant at L138F in fusion protein proved compound 1 to be a RSV fusion inhibitor.

Discovery of Small Molecule Therapeutics for Treatment of Chronic HBV Infection.

The chronic infection of hepatitis B virus (HBV) inflicts 250 million people worldwide representing a major public health threat. A significant subpopulation of patients eventually develop cirrhosis and hepatocellular carcinoma (HCC). Unfortunately, none of the current standard therapies for chronic hepatitis B (CHB) result in a satisfactory clinical cure rate. Driven by a highly unmet medical need, multiple pharmaceutical companies and research institutions have been engaged in drug discovery and development to improve the CHB functional cure rate, defined by sustainable viral suppression and HBsAg clearance after a finite treatment. This Review summarizes the recent advances in the discovery and development of novel anti-HBV small molecules. It is believed that an improved CHB functional cure rate may be accomplished via the combination of molecules with distinct MoAs. Thus, certain molecules may evolve into key components of a suitable combination therapy leading to superior outcome of clinical efficacy in the future.

Discovery and Pre-Clinical Characterization of Third-Generation 4-H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors.

Described herein are the discovery and structure-activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.

Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms.

Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) are promising non-nucleos(t)ide HBV replication inhibitors. HAPs are known to promote core protein mis-assembly, but the molecular mechanism of abnormal assembly is still elusive. Likewise, the assembly status of core protein induced by SBA remains unknown. Here we show that SBA, unlike HAP, does not promote core protein mis-assembly. Interestingly, two reference compounds HAP_R01 and SBA_R01 bind to the same pocket at the dimer-dimer interface in the crystal structures of core protein Y132A hexamer. The striking difference lies in a unique hydrophobic subpocket that is occupied by the thiazole group of HAP_R01, but is unperturbed by SBA_R01. Photoaffinity labeling confirms the HAP_R01 binding pose at the dimer-dimer interface on capsid and suggests a new mechanism of HAP-induced mis-assembly. Based on the common features in crystal structures we predict that T33 mutations generate similar susceptibility changes to both compounds. In contrast, mutations at positions in close contact with HAP-specific groups (P25A, P25S, or V124F) only reduce susceptibility to HAP_R01, but not to SBA_R01. Thus, HAP and SBA are likely to have distinctive resistance profiles. Notably, P25S and V124F substitutions exist in low-abundance quasispecies in treatment-naïve patients, suggesting potential clinical relevance.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Structure-Based Design and Synthesis of Potent Cyclic Peptides Inhibiting the YAP-TEAD Protein-Protein Interaction.

The YAP-TEAD protein-protein interaction (PPI) mediates the oncogenic function of YAP, and inhibitors of this PPI have potential usage in treatment of YAP-involved cancers. Here we report the design and synthesis of potent cyclic peptide inhibitors of the YAP-TEAD interaction. A truncation study of YAP interface 3 peptide identified YAP(84-100) as a weak peptide inhibitor (IC50 = 37 µM), and an alanine scan revealed a beneficial mutation, D94A. Subsequent replacement of a native cation-π interaction with an optimized disulfide bridge for conformational constraint and synergistic effect between macrocyclization and modification at positions 91 and 93 greatly boosted inhibitory activity. Peptide 17 was identified with an IC50 of 25 nM, and the binding affinity (K d = 15 nM) of this 17mer peptide to TEAD1 proved to be stronger than YAP(50-171) (K d = 40 nM).

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists.

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

Discovery of biaryl anthranilides as full agonists for the high affinity niacin receptor.

Biaryl anthranilides are reported as potent and selective full agonists for the high affinity niacin receptor GPR109A. The SAR presented outlines approaches to reduce serum shift and both CYPCYP2C8 and CYP2C9 liabilities, while improving PK and maintaining excellent receptor activity. Compound 2i exhibited good in vivo antilipolytic efficacy while providing a significantly improved therapeutic index over vasodilation (flushing) with respect to niacin in the mouse model.

Unusual effects in the pd-catalyzed asymmetric allylic alkylations: synthesis of chiral chromans.

An examination of earlier reports of poor-to-modest results using Pd-catalyzed asymmetric allylic alkylations (AAA) to effect cyclization to form tetrasubstituted carbons reveals several novel factors that can influence this class of reactions. Thus, carboxylate has a major effect on such cyclizations wherein the ee increases from 14% ee favoring the S with no carboxylate to 84% ee favoring the R enantiomer in the presence of 1 equiv of carboxylate. Changing the double bond geometry from E to Z further increases the ee to 97%. Furthermore, the chiral catalyst that forms the R enantiomer with the E-alkene forms the S enantiomer with the Z alkene. In contrast to trisubstituted alkene substrates, disubstituted ones show a decrease in ee in going from the E to Z alkenes. The role of carboxylate appears to be a ligand to Pd during the catalytic cycle, a previously unsuspected phenomenon since such reactions are generally believed to involve pi-allylpalladium cationic complexes. The dependence upon alkene geometry helps define the nature of the chiral pocket which better accommodates a Z alkene compared to an E alkene. The results are compatible with the enantiodiscriminating step being ionization which occurs by coordination of the palladium to one of the two prochiral faces of the double bond. A synthesis of (+)-clusifoliol, a constituent of a folk medicine for treatment of malignant tumors, which also assigns the absolute configuration, illustrates the utility of the method.