Drug Metabolism And Pharmacokinetics Research Articles

Preclinical metabolism and disposition of [14C]GFH009, a novel selective CDK9 inhibitor

1. GFH009 is a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor currently under phase II clinical trials. In this study, we investigated the metabolism and disposition of GFH009 in Sprague-Dawley (SD) rats, as well as in vitro metabolism of CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human. 2. A radiolabelled study indicated that [14C]GFH009 was quickly and widely distributed throughout the body, but presented low levels in brain, testis and epididymis after a single intravenous dose of 6 mg (100 µCi)/kg to SD rats. 3. GFH009 undergoes systemic metabolic changes, primarily throughO-demethylation, oxidation to carboxylic acid and N-dealkylation, cleavage off the methoxyisopropyl moiety being a minor pathway. These metabolic pathways were found to be mainly consistent both in vitro and in vivo. 4. In SD rats, GFH009 was rapidly and completely eliminated, with feces serving as the major excretion pathway and urine serving as the minor one. Besides, the major clearance pathway for GFH009 was excretion and the minor one was metabolism. 5. GFH009 exhibits favourable drug metabolism and pharmacokinetics (DMPK) properties, which provides valuable insights into the disposition of GFH009 and can be used to guide future clinical studies.

CNSC-31. INVESTIGATING THE THERAPEUTIC POTENTIAL OF STK17A INHIBITION IN GLIOBLASTOMA

Abstract For decades, the survival rate for glioblastoma (GBM) has remained nearly stagnant, with a 5-year survival rate of only 5%. There is an unmet medical need for therapeutic development, because while GBM is the most common primary brain tumor, only four FDA approved drugs have been developed for GBM over the last century. By taking advantage of RNA sequencing, a potential target for GBM has been identified: the kinase STK17A (Serine/Threonine Kinase17A). We confirmed that STK17A is overexpressed in gliomas by analyzing public databases and single-cell RNA sequencing from our own GBM patients, and further associated it with poor patient outcome. Little is known about STK17A, but it has been found to be involved in cell proliferation and tumorigenesis. The role of STK17A in GBM pathophysiology and its therapeutic potential as a drug target remains a critical knowledge gap. To better understand STK17A’s role in GBM pathophysiology, we first knocked down STK17A in GBM cell lines and determined that STK17A had functional roles in proliferation and morphology. Similar conclusions were drawn after removing STK17A in GBM xenograft mouse models. These findings support STK17A as a viable therapeutic target. We then designed and optimized novel STK17A inhibitors for evaluations through kinase enzymatic and biological assays, in vitro proliferation and toxicity assays, and in vivo Drug Metabolism and Pharmacokinetics (DMPK) assays. We then used these potent, selective, and highly brain penetrant STK17A inhibitors to investigate the anti-tumor effects of STK17A inhibition in GBM and demonstrate the efficacy of our novel STK17A inhibitors in vivo. In conclusion, this work seeks to contribute to the field of targeted therapies for GBM by optimizing and examining a novel brain penetrant STK17A inhibitor, initiating studies for the treatment of GBM in vitro and in vivo, and providing proof of efficacy for further studies in GBM patients.

Characterization of Nonclinical Drug Metabolism and Pharmacokinetic Properties of Phosphorodiamidate Morpholino Oligonucleotides, a Novel Drug Class for Duchenne Muscular Dystrophy.

Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0-4.1 h) and golodirsen (2.1-8.7 h) across species and more variable for casimersen (3.2-18.1 h). Plasma protein binding was low (<40%) for all three PMOs in mouse, rat, NHP, and human and was largely concentration independent. In the mdx mouse model of DMD, following a single intravenous injection, extensive biodistribution was observed in the target skeletal muscle tissues and the kidney for all three PMOs; consistent with the latter finding, the predominant route of elimination was renal. In vitro studies using liver microsomes showed no evidence of hepatic metabolism, and none of the PMOs were identified as inhibitors or inducers of the human cytochrome P450 enzymes or membrane drug transporters tested at clinically relevant concentrations. These findings suggest that key DMPK features are consistent for eteplirsen, golodirsen, and casimersen and provide evidence for the concept of a PMO drug class with potential application to novel exon-skipping drug candidates. SIGNIFICANCE STATEMENT: The PMOs eteplirsen, golodirsen, and casimersen share similar absorption, distribution, metabolism, and excretion and DMPK properties, which provides evidence for the concept of a PMO treatment class. A PMO drug class may support a platform approach to enhance understanding of the pharmacokinetic and pharmacodynamic behavior of these molecules. The grouping of novel agent series into platforms could be beneficial in the development of drug candidates for populations in which traditional clinical trials are not feasible.

The Current State of Biotransformation Science - Industry Survey of In Vitro and In Vivo Practices, Clinical Translation, and Future Trends.

Embedded within the field of drug metabolism and pharmacokinetics (DMPK), biotransformation is a discipline that studies the origins, disposition, and structural identity of metabolites to provide a comprehensive safety assessment, including the assessment of exposure coverage in toxicological species. Spanning discovery and development, metabolite identification (metID) scientists employ various strategies and tools to address stage-specific questions aimed at guiding the maturation of early chemical matter into drug candidates. During this process, the identity of major (and minor) circulating human metabolites is ascertained to comply with the regulatory requirements such as the Metabolites in Safety Testing (MIST) guidance. Through the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the "Translatability of MetID In Vitro Systems Working Group" was created within the Translational and ADME Sciences Leadership Group. The remit of this group was to objectively determine how accurate commonly employed in vitro systems have been with respect to prediction of circulating human metabolites, both qualitatively and quantitatively. A survey composed of 34 questions was conducted across 26 pharmaceutical companies to obtain a foundational understanding of current metID practices, preclinically and clinically, as well as to provide perspective on how successful these practices have been at predicting circulating human metabolites.The results of this survey are presented as an initial snapshot of current industry-based metID practices, including our perspective on how a harmonized framework for the conduct of in vitro metID studies could be established. Future perspectives from current practices to emerging advances with greater translational capability are also provided.

Classics in Chemical Neuroscience: Medetomidine.

Medetomidine is an FDA-approved α2-adrenoreceptor (α2-AR) agonist used as a veterinary sedative due to its analgesic, sedative, and anxiolytic properties. While it is marketed for veterinary use as a racemic mixture under the brand name Domitor, the pharmacologically active enantiomer, dexmedetomidine, is approved for sedation and analgesia in the hospital setting. Medetomidine has recently been detected in the illicit drug supply alongside fentanyl, xylazine, cocaine, and heroin, producing pronounced sedative effects that are not reversed by naloxone. The pharmacological effects along with the low cost of supply and lack of regulation for medetomidine has made it a target for misuse. Since 2022, medetomidine has been found as an adulterant in samples of seized drugs, as well as in toxicological analyses of patients admitted to the emergency department after suspected overdoses across several U.S. states and Canada. This Review will discuss the history, chemistry, structure-activity relationships, drug metabolism and pharmacokinetics (DMPK), pharmacology, and emergence of medetomidine as an adulterant in drug mixtures in the context of the current opioid drug crisis.

Discovery of VU6016235: A Highly Selective, Orally Bioavailable, and Structurally Distinct Tricyclic M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM).

Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3-d]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3-d]pyrimidine tricycle core provided lead compound, VU6016235, with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

Improved preclinical drug metabolism and pharmacokinetics of pibothiadine (HEC121210), a novel hepatitis B virus capsid assembly modulator

Pibothiadine (PBD; HEC121120) is a novel hepatitis B virus capsid assembly modulator based on GLS4 (morphothiadine) and has inhibitory activities against resistant strains. To assess the overall preclinical drug metabolism and pharmacokinetics (DMPK) properties of PBD, in vivo pharmacokinetics studies in rats and dogs have been performed along with a series of in vitro metabolism assays. The oral bioavailability of PBD in rats and dogs might be related to its medium permeability in Caco-2 cells and largely be impacted by the pH-dependent solubility. PBD was highly distributed to the liver where the local exposure was 16.4 fold of the system exposure. PBD showed relatively low metabolic rate in recombinant human cytochrome P450 enzymes, whereas low to moderate in vitro clearance in liver microsomes and low (dog) to moderate (rat) in vivo clearance. Furthermore, β-oxidation and dehydrogenation were proposed as the primary metabolic pathways of PBD in rats. Compared to GLS4, the higher systemic exposure of PBD might be attributed to its improved oral absorption and metabolic stability. In addition, the enhanced liver/plasma exposure ratio could further increase the local exposure around the target. These improved DMPK properties might indicate better development of PBD in the clinical phase.

Radioactive ADME Demonstrates ARV-110's High Druggability Despite Low Oral Bioavailability.

Proteolysis-targeting chimeras (PROTACs) have emerged as potentially effective therapeutic medicines, but their high molecular weight and poor solubility directly impact their oral bioavailability. This work synthesized 14C-labeled bavdegalutamide (ARV-110) as a model compound of PROTACs to evaluate its ADME features. Compared with targeted antitumor drugs, the use of food increased oral bioavailability of ARV-110 in rats from 10.75% to 20.97%, which is still undesirable. However, the therapeutic effect of ARV-110 at a low dose was much better than that of enzalutamide, demonstrating the specific catalytic medicinal properties of PROTACs. Moreover, the specific distribution of ARV-110 in subcutaneous prostate tumors was determined by quantitative whole-body autoradiography (QWBA). Notably, the specificity and activity of PROTACs take precedence over their oral absorption, and high oral bioavailability is not necessary to produce excellent therapeutic effects. This work presents a roadmap for developing future PROTAC medications from a radioactive drug metabolism and pharmacokinetics (DMPK) perspective.

Stepwise Structural Simplification of the Dihydroxyanthraquinone Moiety of a Multitarget Rhein-Based Anti-Alzheimer Lead to Improve Drug Metabolism and Pharmacokinetic Properties.

Multitarget compounds have emerged as promising drug candidates to cope with complex multifactorial diseases, like Alzheimer's disease (AD). Most multitarget compounds are designed by linking two pharmacophores through a tether chain (linked hybrids), which results in rather large molecules that are particularly useful to hit targets with large binding cavities, but at the expense of suffering from suboptimal physicochemical/pharmacokinetic properties. Molecular size reduction by removal of superfluous structural elements while retaining the key pharmacophoric motifs may represent a compromise solution to achieve both multitargeting and favorable physicochemical/PK properties. Here, we report the stepwise structural simplification of the dihydroxyanthraquinone moiety of a rhein-huprine hybrid lead by hydroxy group removal-ring contraction-ring opening-ring removal, which has led to new analogs that retain or surpass the potency of the lead on its multiple AD targets while exhibiting more favorable drug metabolism and pharmacokinetic (DMPK) properties and safety profile. In particular, the most simplified acetophenone analog displays dual nanomolar inhibition of human acetylcholinesterase and butyrylcholinesterase (IC50 = 6 nM and 13 nM, respectively), moderately potent inhibition of human BACE-1 (48% inhibition at 15 µM) and Aβ42 and tau aggregation (73% and 68% inhibition, respectively, at 10 µM), favorable in vitro brain permeation, higher aqueous solubility (18 µM) and plasma stability (100/96/86% remaining in human/mouse/rat plasma after 6 h incubation), and lower acute toxicity in a model organism (zebrafish embryos; LC50 >> 100 µM) than the initial lead, thereby confirming the successful lead optimization by structural simplification.

Abstract 3197: Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs)

Abstract Background: SMDCs are gaining momentum with potential advantages over antibody-drug conjugates (ADCs), eg, better tumor penetration and lack of immunogenicity. We have developed a novel platform technology with tailored solutions for ADCs and SMDCs with intracellular and extracellular cleavage options. Key features of the SMDC technology comprise of a small molecule ligand such as an αvβ3 integrin binder showing efficient tumor homing and a linker extracellularly cleaved by neutrophil elastase (NE) in the tumor microenvironment allowing for seamless release of different payload classes.1 Our lead SMDC VIP2362 is currently in a Phase 1 clinical trial for the treatment of advanced solid tumors (NCT05712889). SMDCs often have a shorter half-life compared with ADCs, which may result in more frequent dosing schedules. Here we show significant reduction of clearance and drastic prolongation of half-life of SMDCs achieved by adding a second binding moiety. Methods: Payloads such as kinesin spindle protein inhibitors (KSPi), cyclin-dependent kinase 9 inhibitors (CDK9i), and monomethyl auristatin E (MMAE) have been linked via elastase-cleavable linkers to one or, via a branched linker, to two αvβ3 integrin binding moieties. Cytotoxicity of SMDCs was measured in cell lines with or without NE. Monovalent and bivalent SMDCs were investigated in DMPK studies in rats while monitoring the conjugate and the released payload. Results: Payloads were coupled to the NE-cleavable peptides via different linkages. MMAE was conjugated via sterically hindered ester bonds, CDK9i was conjugated via sulfoximide bonds, and KSPi payloads were coupled via amide bonds. Each of these SMDCs was highly stable in plasma and buffer. Without NE, in vitro cytotoxicity of SMDCs in each cell line was poor with IC50 in the micromolar range. In contrast, in the presence of NE, potency increased, and cytotoxicity (IC50) was in the low nanomolar to picomolar range depending on the cell line and payload. In each case, the SMDCs reached similar potency as the free payload indicating efficient cleavage of the SMDC by NE. In DMPK studies of SMDCs in rat, none or trace levels of free payload (&amp;lt;0.1% as compared to the respective conjugates) was measured. With each of the SMDCs and independent of the conjugated payloads, a remarkable impact on exposure was observed when attaching a second binding ligand. The SMDCs with two ligands showed a 37- to 81-fold reduced plasma clearance as compared with the respective monovalent SMDCs, which parallels a 12- to 57-fold increase in half-life (eg, 2.1 h and 28 h for mono and bivalent MMAE SMDCs respectively). Initial in vivo studies in a cell line-derived xenograft model showed efficacy of bivalent SMDCs with once weekly dosing. Conclusion: Our technology platform comprises NE-cleavable SMDCs with promising options to tune the DMPK profile for a longer half-life. 1. Lerchen et al, AACR (2023). 2. Lerchen et al, Cancers (2023). Citation Format: Anne-Sophie Rebstock, Hans-Georg Lerchen, Harvey Wong, Beatrix Stelte-Ludwig, Mareike Wiedmann, Amy J. Johnson, Raquel Izumi, Ahmed Hamdy. Addressing drug metabolism and pharmacokinetics (DMPK) challenges of small molecule-drug conjugates (SMDCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3197.

Abstract 5931: Investigating the therapeutic potential of STK17A inhibition in GBM

Abstract For decades, the survival rate for glioblastoma (GBM) has remained nearly stagnant, and currently the 5-year survival rate is only 5%. There is an unmet medical need for therapeutic development, as GBM is the most common primary brain tumor, yet only four FDA approved drugs have been developed for GBM over the last century. GBM is notoriously heterogeneous, and this has posed a challenge for researchers seeking to find therapeutic targets. However, by taking advantage of RNA sequencing, a potential biomarker and target for GBM has been identified: the kinase STK17A (Serine/Threonine Kinase 17A), also known as DRAK1. We confirmed that STK17A is overexpressed in brain cancers by analyzing public databases and single-cell RNA sequencing data from GBM patients, and further associated it with poor patient outcome. Little is known about STK17A, but it has been found to be involved with cell proliferation, tumorigenesis, and tumor metastasis. The role of STK17A in GBM pathophysiology and its therapeutic potential as a drug target remains a critical knowledge gap.In order to better understand STK17A’s role in GBM pathophysiology, we first knocked down STK17A in GBM cell lines and determined that STK17A had functional roles in proliferation and morphology. Similar conclusions were drawn after knocking down STK17A in vivo in GBM xenograft mouse models. These findings supported STK17A as a viable therapeutic target. We then designed and optimized novel brain-penetrant STK17A inhibitors with IC50 values of &amp;lt;20nM in enzyme assays and &amp;lt;1μM in cell-based targeting engagement and proliferative assays. The designed and prepared novel STK17A inhibitors were further evaluated through kinase enzymatic assays and biological assays as well as in vitro proliferation and toxicity assays. We further characterized the inhibitors by showing Drug Metabolism and Pharmacokinetics (DMPK) properties in vivo in mouse models at 10 mg/kg via oral administration. We then used these potent, selective, and highly brain penetrant STK17A inhibitors to investigate the anti-tumor effects of STK17A inhibition in GBM in vivo and demonstrate the efficacy of our novel STK17A inhibitors.In conclusion, this work seeks to contribute to the field of targeted therapies for GBM by optimizing and examining the first iterations of novel brain penetrant STK17A inhibitors, initiating studies for the treatment of GBM in vitro and in vivo, and providing proof of efficacy for further studies in GBM patients. Ultimately, the results from our findings will lead to novel strategies for targeted drug development and more robust therapeutic options for GBM patients. Citation Format: Jesus R. Castro. Investigating the therapeutic potential of STK17A inhibition in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5931.

Cytotoxic and Antiproliferative Activity of LASSBio-2208 and the Attempts to Determine Its Drug Metabolism and Pharmacokinetics In Vitro Profile.

Inappropriate expression of histone deacetylase (HDAC-6) and deregulation of the phosphatidylinositol 3-kinase (PI3K) signalling pathway are common aberrations observed in cancers. LASSBio-2208, has been previously described as a dual inhibitor in the nanomolar range of HDAC-6 and PI3Kα and is three times more potent in inhibiting HDAC-6. In this paper we described the cytotoxic and antiproliferative potency of LASSBio-2208 on different tumour cell lines, its possible synergism effect in association with PI3K and HDAC-6 inhibitors, and its drug metabolism and pharmacokinetics (DMPK) in vitro profile. Our studies have demonstrated that LASSBio-2208 has moderate cytotoxic potency on breast cancer cell line MCF-7 (IC50 = 23 µM), human leukaemia cell line CCRF-CEM (IC50 = 8.54 µM) and T lymphoblast cell line MOLT-4 (IC50 = 7.15 µM), with no cytotoxic effect on human peripheral blood mononuclear cells (hPBMC). In addition, it has a good antiproliferative effect on MCF-7 cells (IC50 = 5.44 µM), low absorption by parallel artificial membrane permeability-gastrointestinal tract (PAMPA-GIT) and low permeation by parallel artificial membrane permeability-blood-brain barrier (BBB) (PAMPA-BBB), exhibiting high metabolic stability in rat plasma. Moreover, LASSBio-2208 exhibited synergism when combined with getadolisib and tubastatin A, using the concentrations corresponding to their CC50 values on MOLT-4 and CCRF-CEM cells.

Using Generative Modeling to Endow with Potency Initially Inert Compounds with Good Bioavailability and Low Toxicity.

In the early stages of drug development, large chemical libraries are typically screened to identify compounds of promising potency against the chosen targets. Often, however, the resulting hit compounds tend to have poor drug metabolism and pharmacokinetics (DMPK), with negative developability features that may be difficult to eliminate. Therefore, starting the drug discovery process with a "null library", compounds that have highly desirable DMPK properties but no potency against the chosen targets, could be advantageous. Here, we explore the opportunities offered by machine learning to realize this strategy in the case of the inhibition of α-synuclein aggregation, a process associated with Parkinson's disease. We apply MolDQN, a generative machine learning method, to build an inhibitory activity against α-synuclein aggregation into an initial inactive compound with good DMPK properties. Our results illustrate how generative modeling can be used to endow initially inert compounds with desirable developability properties.

The Comparison of Machine Learning and Mechanistic In Vitro-In Vivo Extrapolation Models for the Prediction of Human Intrinsic Clearance.

Accurate prediction of human pharmacokinetics (PK) remains one of the key objectives of drug metabolism and PK (DMPK) scientists in drug discovery projects. This is typically performed by using in vitro-in vivo extrapolation (IVIVE) based on mechanistic PK models. In recent years, machine learning (ML), with its ability to harness patterns from previous outcomes to predict future events, has gained increased popularity in application to absorption, distribution, metabolism, and excretion (ADME) sciences. This study compares the performance of various ML and mechanistic models for the prediction of human IV clearance for a large (645) set of diverse compounds with literature human IV PK data, as well as measured relevant in vitro end points. ML models were built using multiple approaches for the descriptors: (1) calculated physical properties and structural descriptors based on chemical structure alone (classical QSAR/QSPR); (2) in vitro measured inputs only with no structure-based descriptors (ML IVIVE); and (3) in silico ML IVIVE using in silico model predictions for the in vitro inputs. For the mechanistic models, well-stirred and parallel-tube liver models were considered with and without the use of empirical scaling factors and with and without renal clearance. The best ML model for the prediction of in vivo human intrinsic clearance (CLint) was an in vitro ML IVIVE model using only six in vitro inputs with an average absolute fold error (AAFE) of 2.5. The best mechanistic model used the parallel-tube liver model, with empirical scaling factors resulting in an AAFE of 2.8. The corresponding mechanistic model with full in silico inputs achieved an AAFE of 3.3. These relative performances of the models were confirmed with the prediction of 16 Pfizer drug candidates that were not part of the original data set. Results show that ML IVIVE models are comparable to or superior to their best mechanistic counterparts. We also show that ML IVIVE models can be used to derive insights into factors for the improvement of mechanistic PK prediction.

Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. IMPORTANCE The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

DruMAP: A Novel Drug Metabolism and Pharmacokinetics Analysis Platform.

We developed a novel drug metabolism and pharmacokinetics (DMPK) analysis platform named DruMAP. This platform consists of a database for DMPK parameters and programs that can predict many DMPK parameters based on the chemical structure of a compound. The DruMAP database includes curated DMPK parameters from public sources and in-house experimental data obtained under standardized conditions; it also stores predicted DMPK parameters produced by our prediction programs. Users can predict several DMPK parameters simultaneously for novel compounds not found in the database. Furthermore, the highly flexible search system enables users to search for compounds as they desire. The current version of DruMAP comprises more than 30,000 chemical compounds, about 40,000 activity values (collected from public databases and in-house data), and about 600,000 predicted values. Our platform provides a simple tool for searching and predicting DMPK parameters and is expected to contribute to the acceleration of new drug development. DruMAP can be freely accessed at: https://drumap.nibiohn.go.jp/.

Discovery of quinazolin-4-one-based non-covalent inhibitors targeting the severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 Mpro)

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a great threat to public health while various vaccines are available worldwide. Main protease (Mpro) has been validated as an effective anti-COVID-19 drug target. Using medicinal chemistry and rational drug design strategies, we identified a quinazolin-4-one series of nonpeptidic, noncovalent SARS-CoV-2 Mpro inhibitors based on baicalein, 5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one. In particular, compound C7 exhibits superior inhibitory activity against SARS-CoV-2 Mpro relative to baicalein (IC50 = 0.085 ± 0.006 and 0.966 ± 0.065 μM, respectively), as well as improved physicochemical and drug metabolism and pharmacokinetics (DMPK) properties. In addition, C7 inhibits viral replication in SARS-CoV-2-infected Vero E6 cells more effectively than baicalein (EC50 = 1.10 ± 0.12 and 5.15 ± 1.64 μM, respectively) with low cytotoxicity (CC50 ＞ 50 μM). An X-ray co-crystal structure reveals a non-covalent mechanism of action, and a noncanonical binding mode not observed by baicalein. These results suggest that C7 represents a promising lead for development of more effective SARS-CoV-2 Mpro inhibitors and anti-COVID-19 drugs.

METTL7A (TMT1A) and METTL7B (TMT1B) Are Responsible for Alkyl S-Thiol Methyl Transferase Activity in Liver.

S-methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine (SAM)-dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine prodrugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol methyltransferase. METTL7B is an endoplasmic reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM-dependent thiol methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC50 = 1.17 μM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed thiol methyltransferase 1A (TMT1A) and thiol methyltransferase 1B (TMT1B), respectively, that are responsible for thiol methylation activity in human liver microsomes. SIGNIFICANCE STATEMENT: We identified methyltransferase-like protein 7A (thiol methyltransferase 1A) and methyltransferase-like protein 7B (thiol methyltransferase 1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S-methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible for this activity will improve our understanding of the drug metabolism and pharmacokinetic (DMPK) properties of alkyl- or phenolic thiol-containing therapeutics.

Design, synthesis, and pharmacological evaluation of N-(3-carbamoyl-1H-pyrazol-4-yl)-1,3-oxazole-4-carboxamide derivatives as interleukin-1 receptor-associated kinase 4 inhibitors with reduced potential for cytochrome P450 1A2 induction

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical molecule in Toll-like receptor/interleukin-1 receptor signaling and an attractive therapeutic target for a wide range of inflammatory and autoimmune diseases as well as cancers. In our search for novel IRAK4 inhibitors, we conducted structural modification of a thiazolecarboxamide derivative 1, a lead compound derived from high-throughput screening hits, to elucidate structure–activity relationship and improve drug metabolism and pharmacokinetic (DMPK) properties. First, conversion of the thiazole ring of 1 to an oxazole ring along with introduction of a methyl group at the 2-position of the pyridine ring aimed at reducing cytochrome P450 (CYP) inhibition were conducted to afford 16. Next, modification of the alkyl substituent at the 1-position of the pyrazole ring of 16 aimed at improving CYP1A2 induction properties revealed that branched alkyl and analogous substituents such as isobutyl (18) and (oxolan-3-yl)methyl (21), as well as six-membered saturated heterocyclic groups such as oxan-4-yl (2), piperidin-4-yl (24, 25), and dioxothian-4-y (26), are effective for reducing induction potential. Representative compound AS2444697 (2) exhibited potent IRAK4 inhibitory activity with an IC50 value of 20 nM and favorable DMPK properties such as low risk of drug–drug interactions mediated by CYPs as well as excellent metabolic stability and oral bioavailability.

Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents.

Identification of structurally novel inhibitors of lysine methyltransferase G9a has been a subject of intense research in cancer epigenetics. Starting with the high-throughput screening (HTS) hit rac-10a obtained from the chemical library of the University of Tokyo Drug Discovery Initiative, the structure-activity relationship of the unique substrate-competitive inhibitors was established with the help of X-ray crystallography and fragment molecular orbital (FMO) calculations for the ligand-protein interaction. Further optimization of the in vitro characteristics and drug metabolism and pharmacokinetics (DMPK) properties led to the identification of 26j (RK-701), which is a structurally distinct potent inhibitor of G9a/GLP (IC50 = 27/53 nM). Compound 26j exhibited remarkable selectivity against other related methyltransferases, dose-dependent attenuation of cellular H3K9me2 levels, and tumor growth inhibition in MOLT-4 cells in vitro. Moreover, compound 26j showed inhibition of tumor initiation and growth in a carcinogen-induced hepatocellular carcinoma (HCC) in vivo mouse model without overt acute toxicity.