Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Discovery of DS68702229 as a Potent, Orally Available NAMPT (Nicotinamide Phosphoribosyltransferase) Activator.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) salvage pathway. Because NAD+ plays a pivotal role in energy metabolism and boosting NAD+ has positive effects on metabolic regulation, activation of NAMPT is an attractive therapeutic approach for the treatment of various diseases, including type 2 diabetes and obesity. Herein we report the discovery of 1-(2-phenyl-1,3-benzoxazol-6-yl)-3-(pyridin-4-ylmethyl)urea 12c (DS68702229), which was identified as a potent NAMPT activator. Compound 12c activated NAMPT, increased cellular NAD+ levels, and exhibited an excellent pharmacokinetic profile in mice after oral administration. Oral administration of compound 12c to high-fat diet-induced obese mice decreased body weight. These observations indicate that compound 12c is a promising anti-obesity drug candidate.

Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies.

Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

Discovery of Potent Antiallergic Agents Based on an o-Aminopyridinyl Alkynyl Scaffold.

Effective therapeutic agents are highly desired for immune-mediated allergic diseases. Herein, we report the design, synthesis, and structure-activity relationship of an o-aminopyridinyl alkyne series as novel orally bioavailable antiallergic agents, which was identified through phenotypic screening. Compound optimization yielded a highly potent compound 36, which effectively suppressed mast cell degranulation in a dose-dependent manner (IC50, 2.54 nM for RBL-2H3 cells; 48.28 nM for peritoneal mast cells (PMCs)) with a good therapeutic index. It also regulated the activation of FcεRI-mediated downstream signaling proteins in IgE/Ag-stimulated RBL-2H3 cells. In addition, 36 exhibited excellent in vivo pharmacokinetic properties and antiallergic efficacy in both passive systemic anaphylaxis (PSA) and house dust mite (HDM)-induced murine models of pulmonary allergic inflammation. Furthermore, preliminary analysis of the kinases profile identified Src-family kinases as potential targets for 36. Compound 36 may serve as a new valuable lead compound for future antiallergic drug discovery.

Potent inhibition of arenavirus infection by a novel fusion inhibitor.

Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.

Small Molecules of Marine Origin as Potential Anti-Glioma Agents.

Marine organisms are able to produce a plethora of small molecules with novel chemical structures and potent biological properties, being a fertile source for discovery of pharmacologically active compounds, already with several marine-derived agents approved as drugs. Glioma is classified by the WHO as the most common and aggressive form of tumor on CNS. Currently, Temozolomide is the only chemotherapeutic option approved by the FDA even though having some limitations. This review presents, for the first time, a comprehensive overview of marine compounds described as anti-glioma agents in the last decade. Nearly fifty compounds were compiled in this document and organized accordingly to their marine sources. Highlights on the mechanism of action and ADME properties were included. Some of these marine compounds could be promising leads for the discovery of new therapeutic alternatives for glioma treatment.

Intestinal Excretion, Intestinal Recirculation, and Renal Tubule Reabsorption Are Underappreciated Mechanisms That Drive the Distribution and Pharmacokinetic Behavior of Small Molecule Drugs.

Drug reabsorption following biliary excretion is well-known as enterohepatic recirculation (EHR). Renal tubular reabsorption (RTR) following renal excretion is also common but not easily assessed. Intestinal excretion (IE) and enteroenteric recirculation (EER) have not been recognized as common disposition mechanisms for metabolically stable and permeable drugs. IE and intestinal reabsorption (IR:EHR/EER), as well as RTR, are governed by dug concentration gradients, passive diffusion, active transport, and metabolism, and together they markedly impact disposition and pharmacokinetics (PK) of small molecule drugs. Disruption of IE, IR, or RTR through applications of active charcoal (AC), transporter knockout (KO), and transporter inhibitors can lead to changes in PK parameters. The impacts of intestinal and renal reabsorption on PK are under-appreciated. Although IE and EER/RTR can be an intrinsic drug property, there is no apparent strategy to optimize compounds based on this property. This review seeks to improve understanding and applications of IE, IR, and RTR mechanisms.

Protective effects of a small molecule inhibitor, DDQ against amyloid beta in Alzheimer's disease.

The purpose of our study is to determine the protective effects of the newly discovered molecule DDQ (diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl) methylphosphonate) against mutant APP and amyloid-beta (Aß) in Alzheimer's disease (AD). To achieve our objective, we used a well characterized amyloid-beta precursor protein (APP) transgenic mouse model (Tg2576 strain). We administered DDQ, a 20 mg/kg body weight (previously determined in our laboratory) intra-peritoneally 3-times per week for 2 months, starting at the beginning of the 12th month, until the end of the 14th month. Further, using biochemical and molecular methods, we measured the levels of DDQ in the blood, skeletal muscle, and brain. Using Morris Water Maze, Y-maze, open field, and rotarod tests, we assessed cognitive behavior after DDQ treatment. Using q-RT-PCR, immunoblotting, transmission electron microscopy, and Golgi-cox staining methods, we studied mRNA and protein levels of longevity genes SIRTUINS, mitochondrial number & length, and dendritic spine number and length in DDQ-treated APP mice. Our extensive pharmacodynamics analysis revealed high peak levels of DDQ in the skeletal muscle, followed by serum and brain. Our behavioral analysis of rotarod, open field, Y-maze, and Morris Water Maze tests revealed that DDQ ameliorated cognitive decline (Morris Water Maze), improved working memory (Y-Maze), exploratory behavior (open field), and motor coordination (rotarod) in DDQ-treated APP mice. Interestingly, longevity genes SIRTUINS, mitochondrial biogenesis, fusion, mitophagy, autophagy and synaptic genes were upregulated in DDQ-treated APP mice relative to untreated APP mice. Dendritic spines and the quality mitochondria were significantly increased in DDQ treated APP mice. Current study findings, together with our previous study observations, strongly suggest that DDQ has anti-aging, and anti-amyloid-beta effects and a promising molecule to reduce age-and amyloid-beta-induced toxicities in AD.

Drugs, Metabolites, and Lung Accumulating Small Lysosomotropic Molecules: Multiple Targeting Impedes SARS-CoV-2 Infection and Progress to COVID-19.

Lysosomotropism is a biological characteristic of small molecules, independently present of their intrinsic pharmacological effects. Lysosomotropic compounds, in general, affect various targets, such as lipid second messengers originating from lysosomal enzymes promoting endothelial stress response in systemic inflammation; inflammatory messengers, such as IL-6; and cathepsin L-dependent viral entry into host cells. This heterogeneous group of drugs and active metabolites comprise various promising candidates with more favorable drug profiles than initially considered (hydroxy) chloroquine in prophylaxis and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections/Coronavirus disease 2019 (COVID-19) and cytokine release syndrome (CRS) triggered by bacterial or viral infections. In this hypothesis, we discuss the possible relationships among lysosomotropism, enrichment in lysosomes of pulmonary tissue, SARS-CoV-2 infection, and transition to COVID-19. Moreover, we deduce further suitable approved drugs and active metabolites based with a more favorable drug profile on rational eligibility criteria, including readily available over-the-counter (OTC) drugs. Benefits to patients already receiving lysosomotropic drugs for other pre-existing conditions underline their vital clinical relevance in the current SARS-CoV2/COVID-19 pandemic.

Safety and pharmacokinetics of milademetan, a MDM2 inhibitor, in Japanese patients with solid tumors: A phase I study.

Milademetan (DS-3032, RAIN-32) is an orally available mouse double minute 2 (MDM2) antagonist with potential antineoplastic activity owing to increase in p53 activity through interruption of the MDM2-p53 interaction. This phase I, dose-escalating study assessed the safety, tolerability, efficacy, and pharmacokinetics of milademetan in 18 Japanese patients with solid tumors who relapsed after or were refractory to standard therapy. Patients aged ≥ 20 years received oral milademetan once daily (60 mg, n = 3; 90 mg, n = 11; or 120 mg, n = 4) on days 1 to 21 in a 28-day cycle. Dose-limiting toxicities, safety, tolerability, maximum tolerated dose, pharmacokinetics, and recommended dose for phase II were determined. The most frequent treatment-emergent adverse events included nausea (72.2%), decreased appetite (61.1%), platelet count decreased (61.1%), white blood cell count decreased (50.0%), fatigue (50.0%), and anemia (50.0%). Dose-limiting toxicities (three events of platelet count decreased and one nausea) were observed in the 120-mg cohort. The plasma concentrations of milademetan increased in a dose-dependent manner. Stable disease was observed in seven out of 16 patients (43.8%). Milademetan was well tolerated and showed modest antitumor activity in Japanese patients with solid tumors. The recommended dose for phase II was considered to be 90 mg in the once-daily 21/28-day schedule. Future studies would be needed to further evaluate the potential safety, tolerability, and clinical activity of milademetan in patients with solid tumors and lymphomas. The trial was registered with Clinicaltrials.jp: JapicCTI-142693.

Design of Thioether Cyclic Peptide Scaffolds with Passive Permeability and Oral Exposure.

Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel but independent tracks. Less precedent combines their respective attributes, thereby limiting the potential to identify permeable peptide ligands for key targets. Herein, we present novel 6-, 7-, and 8-mer cyclic peptides (MW 774-1076 g·mol-1) with passive permeability and oral exposure that feature the amino acids and thioether ring-closing common to large array formats, including DNA- and RNA-templated synthesis. Each oral peptide herein, selected from virtual libraries of partially N-methylated peptides using in silico methods, reflects the subset consistent with low energy conformations, low desolvation penalties, and passive permeability. We envision that, by retaining the backbone N-methylation pattern and consequent bias toward permeability, one can generate large peptide arrays with sufficient side chain diversity to identify permeability-biased ligands to a variety of protein targets.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

The Vital Role of Proteomics in Characterizing Novel Protein Degraders.

Mass spectrometry-based proteomics profiling is a discovery tool that enables researchers to understand the mechanisms of action of drug candidates. When applied to proteolysis targeting chimeras (PROTACs) such approaches provide unbiased perspectives of the binding, degradation selectivity, and mechanism related to efficacy and safety. Specifically, global profiling experiments can identify direct degradation events and assess downstream pathway modulation that may result from degradation or off-target inhibition. Targeted proteomics approaches can be used to quantify the levels of relevant E3 ligases and the protein of interest in cell lines and tissues of interest, which can inform the line of sight and provide insights on possible safety liabilities early in the project. Furthermore, proteomics approaches can be applied to understand protein turnover and resynthesis rates and inform on target tractability, as well as pharmacokinetics/pharmacodynamics understanding. In this perspective, we survey the literature around the impact of mass spectrometry-based proteomics in the development of PROTACs and present our envisioned proteomics cascade for supporting targeted protein degradation projects.

Discovery of Highly Potent Fusion Inhibitors with Potential Pan-Coronavirus Activity That Effectively Inhibit Major COVID-19 Variants of Concern (VOCs) in Pseudovirus-Based Assays.

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

Pyrimido[1,2-b]indazole derivatives: Selective inhibitors of human monoamine oxidase B with neuroprotective activity.

Structurally diverse heterotricyclic compounds are recognized as monoamine oxidase (MAO) inhibitors and thus represent an appealing scaffold in development and optimization of novel MAO inhibitors. Herein we explored the chemical space of pyrimido[1,2-b]indazoles as MAO inhibitors by preparing a small library of (hetero)aryl derivatives. An efficient synthetic strategy was developed starting from commercially available 1H-indazol-3-amines, which were converted to various 3-bromoheterotricyclic derivatives and further functionalized via Suzuki-Miyaura coupling reaction. Derivatives 4a-t selectively inhibited human MAO-B isoform in a reversible and competitive manner as confirmed by kinetic experiments and docking studies. Selected derivatives were not cytotoxic to neuroblastoma SH-SY5Y cells. Moreover, analogue 4i protected human neuroblastoma SH-SY5Y cells against 6-hydroxydopamine-induced cell death, which confirms the applicability of the pyrimido[1,2-b]indazoles as potential antiparkinsonian agents.

A Phase 1 study of RO6870810, a novel bromodomain and extra-terminal protein inhibitor, in patients with NUT carcinoma, other solid tumours, or diffuse large B-cell lymphoma.

BACKGROUND: Bromodomain and extra-terminal (BET) proteins are epigenetic readers that can drive carcinogenesis and therapy resistance. RO6870810 is a novel, small-molecule BET inhibitor. METHODS: We conducted a Phase 1 study of RO6870810 administered subcutaneously for 21 or 14 days of 28- or 21-day cycles, respectively, in patients with the nuclear protein of the testis carcinoma (NC), other solid tumours, or diffuse large B-cell lymphoma (DLBCL) with MYC deregulation. RESULTS: Fatigue (42%), decreased appetite (35%) and injection-site erythema (35%) were the most common treatment-related adverse events. Pharmacokinetic parameters demonstrated linearity over the dose range tested and support once-daily dosing. Pharmacodynamic assessments demonstrated sustained decreases in CD11b levels in peripheral blood mononuclear cells. Objective response rates were 25% (2/8), 2% (1/47) and 11% (2/19) for patients with NC, other solid tumours and DLBCL, respectively. Responding tumours had evidence of deregulated MYC expression. CONCLUSIONS: This trial establishes the safety, favourable pharmacokinetics, evidence of target engagement and preliminary single-agent activity of RO6870810. Responses in patients with NC, other solid tumours and DLBCL provide proof-of-principle for BET inhibition in MYC-driven cancers. The results support further exploration of RO6870810 as monotherapy and in combinations. CLINICAL TRIALS REGISTRATION: NCT01987362.

An Integrated Pharmacophore/Docking/3D-QSAR Approach to Screening a Large Library of Products in Search of Future Botulinum Neurotoxin A Inhibitors.

Botulinum toxins are neurotoxins produced by Clostridium botulinum. This toxin can be lethal for humans as a cause of botulism; however, in small doses, the same toxin is used to treat different conditions. Even if the therapeutic doses are effective and safe, the adverse reactions could be local and could unmask a subclinical impairment of neuromuscular transmissions. There are not many cases of adverse events in the literature; however, it is possible that sometimes they do not occur as they are transient and, if they do occur, there is no possibility of a cure other than to wait for the pharmacological effect to end. Inhibition of botulinum neurotoxin type A (BoNT/A) effects is a strategy for treating botulism as it can provide an effective post-exposure remedy. In this paper, 13,592,287 compounds were screened through a pharmacophore filter, a 3D-QSAR model, and a virtual screening; then, the compounds with the best affinity were selected. Molecular dynamics simulation studies on the first four compounds predicted to be the most active were conducted to verify that the poses foreseen by the docking were stable. This approach allowed us to identify compounds with a calculated inhibitory activity in the range of 316-500 nM.

Selective Inhibitors of G2019S-LRRK2 Kinase Activity.

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.