De novo design of SARS-CoV-2 main protease inhibitors with characteristic binding modes.

The coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spreads rapidly all over the world. The main protease (Mpro) is significant to the replication and transcription of viruses, making it an attractive drug target against coronaviruses. Here, we introduce a series of novel inhibitors which are designed de novo through structure-based drug design approach that have great potential to inhibit SARS-CoV-2 Mproin vitro. High-resolution structures show that these inhibitors form covalent bonds with the catalytic cysteine through the novel dibromomethyl ketone (DBMK) as a reactive warhead. At the same time, the designed phenyl group beside the DBMK warhead inserts into the cleft between H41 and C145 through π-π stacking interaction, splitting the catalytic dyad and disrupting proton transfer. This unique binding model provides novel clues for the cysteine protease inhibitor development of SARS-CoV-2 as well as other pathogens.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Triazine-Based Covalent DNA-Encoded Libraries for Discovery of Covalent Inhibitors of Target Proteins.

Since ibrutinib was approved by the FDA as an effective monotherapy for chronic lymphocytic leukemia (CLL) and multilymphoma, more and more FDA-approved covalent drugs are coming back into the market. On this occasion, the resurgence of interest in covalent drugs calls for more hit discovery techniques. However, the limited numbers of covalent libraries prevent the development of this area. Herein, we report the design of covalent DNA-encoded library (DEL) and its selection method for the discovery of covalent inhibitors for target proteins. These triazine-based covalent DELs yielded potent compounds after covalent selection against target proteins, including Bruton's Tyrosine Kinase (BTK), Janus kinase 3 (JAK3), and peptidyl-prolyl cis/trans isomerase NIMA-interacting-1 (Pin1).

Structure-Activity Relationship Study of Indolin-5-yl-cyclopropanamine Derivatives as Selective Lysine Specific Demethylase 1 (LSD1) Inhibitors.

LSD1 is identified as an essential drug target, which is closely correlated to the development of several tumor types. In this work, on the basis of comprehensive analysis of the binding site of LSD1 and other FAD-dependent enzymes, a novel series of potent and selective LSD1 inhibitors were designed by incorporation of privileged indoline scaffold strategies. Representative compound 7e (LSD1; IC50 = 24.43 nM, selectivity over LSD2 and MAOs of >200- and 4000-fold) possessed selective antiproliferative activities against MV-4-11 cell lines. Further study indicates that 7e could activate CD86 expression (EC50 = 470 nM) and induce differentiation of AML cell lines. More importantly, compound 7e demonstrated an acceptable oral PK profile and good in vivo antitumor efficacy with a T/C value of 30.89% in an MV-4-11 xenograft mouse model. Collectively, this work provides a promising lead compound for the development of novel LSD1 inhibitors for the treatment of AML.

Distal mutation V486M disrupts the catalytic activity of DPP4 by affecting the flap of the propeller domain.

Dipeptidyl peptidase-4 (DPP4) plays a crucial role in regulating the bioactivity of glucagon-like peptide-1 (GLP-1) that enhances insulin secretion and pancreatic ß-cell proliferation, making it a therapeutic target for type 2 diabetes. Although the crystal structure of DPP4 has been determined, its structure-function mechanism is largely unknown. Here, we examined the biochemical properties of sporadic human DPP4 mutations distal from its catalytic site, among which V486M ablates DPP4 dimerization and causes loss of enzymatic activity. Unbiased molecular dynamics simulations revealed that the distal V486M mutation induces a local conformational collapse in a ß-propeller loop (residues 234-260, defined as the flap) and disrupts the dimerization of DPP4. The "open/closed" conformational transitions of the flap whereby capping the active site, are involved in the enzymatic activity of DPP4. Further site-directed mutagenesis guided by theoretical predictions verified the importance of the conformational dynamics of the flap for the enzymatic activity of DPP4. Therefore, the current studies that combined theoretical modeling and experimental identification, provide important insights into the biological function of DPP4 and allow for the evaluation of directed DPP4 genetic mutations before initiating clinical applications and drug development.

Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma.

HDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%-35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

Design, Synthesis, and Biological Evaluation of HDAC Degraders with CRBN E3 Ligase Ligands.

Histone deacetylases (HDACs) play important roles in cell growth, cell differentiation, cell apoptosis, and many other cellular processes. The inhibition of different classes of HDACs has been shown to be closely related to the therapy of cancers and other diseases. In this study, a series of novel CRBN-recruiting HDAC PROTACs were designed and synthesized by linking hydroxamic acid and benzamide with lenalidomide, pomalidomide, and CC-220 through linkers of different lengths and types. One of these PROTACs, denoted 21a, with a new benzyl alcohol linker, exhibited comparably excellent HDAC inhibition activity on different HDAC classes, acceptable degradative activity, and even better in vitro anti-proliferative activities on the MM.1S cell line compared with SAHA. Moreover, we report for the first time the benzyl alcohol linker, which could also offer the potential to be used to develop more types of potent PROTACs for targeting more proteins of interest (POI).

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma.

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2 inhibitors. Among all the prepared compounds, 9c displayed a good NSD2 inhibitory activity (IC50 = 2.7 µM) and selectivity against both SET-domain-containing and non-SET-domain-containing methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by significantly suppressed the methylation of H3K36me2. Compound 9c specifically inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of NSD2-targeted genes. When administered intraperitoneally at 25 mg/kg, compound 9c suppressed the tumor growth of RS4:11 xenografts in vivo and no body weight loss was detected in the tested SCID mice.

Discovery of a Potent and Selective NF-κB-Inducing Kinase (NIK) Inhibitor That Has Anti-inflammatory Effects in Vitro and in Vivo.

The overexpression of NIK plays a critical role in liver inflammatory diseases. Treatment of such diseases with small-molecule NIK inhibitors is a reasonable but underexplored approach. In this paper, we reported the discovery of a potent and selective NIK inhibitor 46 (XT2). 46 inhibited the NIK kinase with an IC50 value of 9.1 nM in vitro, and it also potently suppressed NIK activities in intact cells. In isogenic primary hepatocytes, treatment of 46 efficiently suppressed the expressions of NIK-induced genes. 46 was orally bioavailable in mice with moderate systemic exposure. In a NIK-associated mouse liver inflammation model, 46 suppressed CCl4-induced upregulation of ALT, a key biomarker of acute liver injury. 46 also decreased immune cell infiltration into the injured liver tissue. Overall, these studies provide examples that an NIK inhibitor is able to suppress toxin-induced liver inflammations, which indicates its therapeutic potentials for the treatment of liver inflammatory diseases.

Synthesis and in Vitro and in Vivo Biological Evaluation of Tissue-Specific Bisthiazole Histone Deacetylase (HDAC) Inhibitors.

A series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors was developed during our previous work. In the present work, a new series of highly potent bisthiazole-based compounds were designed and synthesized. Among the prepared compounds, compound H13, which contains an α-(S)-methyl-substituted benzyl group, displays potent inhibitory activity toward human HDACs and several cancer cells lines. Compound H13 has a favorable PK profile and high tissue distribution specificity in the colon, as well as good efficacy in the AOM-DSS mouse model for colitis-associated colonic tumorigenesis.

Tranylcypromine and 6-trifluoroethyl thienopyrimidine hybrid as LSD1 inhibitor.

Tranylcypromine moiety extracted from LSD1 inhibitors and 6-trifluoroethyl thienopyrimidine moiety from menin-MLL1 PPI inhibitors were merged to give new chemotypes for medicinal chemistry study. Among 15 new compounds prepared in this work, some exhibited nanomolar LSD1 activity and good selectivity over MAO-A/B, low micromolar menin-MLL1 PPI inhibitory activity, as well as submicromolar MV4-11 antiprofilative activities. Intracellular LSD1 engagement of compounds with higher enzymatic and antiproliferative activities was confirmed by CD86 mRNA up-regulation experiments.

Discovery of a Natural-Product-Derived Preclinical Candidate for Once-Weekly Treatment of Type 2 Diabetes.

Poor medication adherence is one of the leading causes of suboptimal glycaemic control in approximately half of the patients with type 2 diabetes mellitus (T2DM). Long-acting antidiabetic drugs are clinically needed for improving patients' compliance. Dipeptidyl peptidase-4 (DPP-4) inhibitors play an increasingly important role in the treatment of T2DM because of their favorable properties of weight neutrality and hypoglycemia avoidance. Herein, we report the successful discovery and scale-up synthesis of compound 5, a structurally novel, potent, and long-acting DPP-4 inhibitor for the once-weekly treatment of T2DM. Inhibitor 5 has fast-associating and slow-dissociating binding kinetics profiles as well as slow clearance rate and long terminal half-life pharmacokinetic properties. A single-dose oral administration of 5 (3 mg/kg) inhibited >80% of DPP-4 activity for more than 7 days in diabetic mice. The long-term antidiabetic efficacies of 5 (10 mg/kg, qw) were better than those of the once-weekly trelagliptin and omarigliptin, especially in decreasing the hemoglobin A1c level.

A series of camptothecin prodrugs exhibit HDAC inhibition activity.

Camptothecin plays an important role in clinical cancer treatment, and its derivatives are a favorite of pharmaceutical chemists. Herein, we have designed a series of camptothecin prodrugs that exhibit histone deacetylase (HDAC) inhibition activity based on the synergy effect between HDAC inhibitors and camptothecin derivatives. With the evaluation of stability in buffers or plasma from human or mouse model, an appropriate linker was found, so the active drug can be released efficiently and compound 21a exhibited strong antiproliferative activity in A549 and HCT-116 cell lines. These results indicated that the well-designed prodrug can be promising in cancer treatment.

Clinical and Physiological Characterization of Elevated Plasma Glucagon-Like Peptide-1 Levels (Hyperglipemia) in a Dipeptidyl Peptidase IV Mutation Carrier.

The clinical application of dipeptidyl peptidase IV inhibitors (DPP4i) increasing active glucagon-like peptide-1 (AGLP-1) levels has been linked to pancreatitis, pancreatic tumors, and cardiovascular events. However, DPP4 mutations in humans or the long-term outcomes of high glucagon-like peptide-1 (GLP-1) level exposure have not been reported. A trio family with a proband showing an extremely high AGLP-1 level [defined here as hyperglipemia (hyper-glucagon-like peptide-1-emia)] were conducted whole-exome sequencing for potential pathogenic genetic defects. One novel DPP4 mutation, p.V486M (c.1456 G>A), was identified in the proband and showed damaged enzymatic activity of DPP4. Ex vivo functional study further showed that the serum from the proband markedly enhanced insulin production of primary rat islet cells. Furthermore, V486M variant and another eight DPP4 variants were identified in our in-home database and seven showed decreased enzymatic activities than wild-type DPP4, consistent with their alterations in their protein expression levels. Of note, the levels of glucose, lipids, and tumor markers (especially for CA15-3 and CA125), increased gradually in the proband during a 4-year follow-up period, although no abnormal physical symptoms or imaging results were observed at present. The other two old carriers in the pedigree both had type 2 diabetes, and one of them also had hyperlipidemia and myocarditis. We first identified hyperglipemia in a female subject harboring a loss-of-function DPP4 mutation with decreased DPP4 activity. Other sporadic DPP4 mutations verified the low-frequent occurrence of genetic inhibition of DPP4 activity, at least in the Chinese population studied. These results may provide new evidence for evaluation of the potential long-term effects of DPP4i and GLP-1 analogs.

Novel spirocyclic tranylcypromine derivatives as lysine-specific demethylase 1 (LSD1) inhibitors.

Herein we describe the design, synthesis, and biological evaluation of a novel series of tranylcypromine-based LSD1 inhibitors via conformational restriction using spiro ring systems. A simple, direct spirocyclic analog of tranylcypromine (compounds 8a and 8b) was shown to be a 28- to 129-fold more potent inhibitor of LSD1 enzyme compared to tranylcypromine. Further incorporation of various substituted benzyl groups to the amino group resulted in a suite of 2',3'-dihydrospiro[cyclopropane-1,1'-inden]-2-amines that are potent LSD1 inhibitors with excellent selectivity profiles (e.g.14a, 15b, 16a, 19a and 20b) against closely related enzymes such as MAO-A, MAO-B, and LSD2.

Tying up tranylcypromine: Novel selective histone lysine specific demethylase 1 (LSD1) inhibitors.

Aberrant expression of lysine specific histone demethylase 1 (LSD1) has been increasingly associated with numerous cancer cells and several proof-of-concept studies are strongly suggestive of its potential as a druggable target. Tranylcypromine (TCP) is an antidepressant originally known to target the monoamine oxidases A and B (MAO-A and MAO-B), which are structurally related to LSD1. A number of TCP derivatives have been identified as potent LSD1 inhibitors, with a handful of them currently being tested in clinical trials. However, thus far the majority of structure-activity relationship studies reported on these TCP derivatives have been mostly limited to the racemates. In this study, we present the SAR data for a novel series of conformationally-restricted TCP-based LSD1 inhibitors, both in their racemic and enantiomerically pure forms. Compounds 18b and 19b were identified as the most potent LSD1 inhibitors within this series, possessing excellent selectivity (>10,000-fold) against MAO-A and MAO-B. These compounds activated CD86 expression on the human MV4-11 AML cells following 10 days of exposure, accompanied with the apparent cytotoxicity. Taken together, these findings are consistent with the pharmacological inhibition of LSD1 and further provide structural insights on the binding modes of these TCP derivatives and their enantiomers at the LSD1.

Design, synthesis and biological evaluation of thienopyrimidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.

New thienopyrimidine hydroxamic acid derivatives as HDACs inhibitors were designed, synthesized and evaluated. All compounds were evaluated for their ability to inhibit recombinant human HDAC1, HDAC3, and HDAC6 isoforms and in vitro anti-proliferative activity on tumor cell lines RMPI 8226 and HCT 116. Most of these compounds displayed good to excellent inhibitory activities against HDACs. The IC50 values of compound 9m against HDAC1, HDAC3, and HDAC6 was 29.81 ± 0.52 nM, 24.71 ± 1.16 nM, and 21.29 ± 0.32 nM. Most of these compounds showed strong anti-proliferative activity against human cancer cell lines including RMPI 8226 and HCT 116. The IC50 values of compound 9m against RPMI 8226 and HCT 116 proliferation were 0.97 ± 0.072 µM and 1.01 ± 0.033 µM, respectively. In addition, compound 9m noticeably up-regulated the level of histone H3 acetylation at the low concentration of 0.3 µM.

Discovery and Rational Design of Natural-Product-Derived 2-Phenyl-3,4-dihydro-2H-benzo[f]chromen-3-amine Analogs as Novel and Potent Dipeptidyl Peptidase 4 (DPP-4) Inhibitors for the Treatment of Type 2 Diabetes.

Starting from the lead isodaphnetin, a natural product inhibitor of DPP-4 discovered through a target fishing docking based approach, a series of novel 2-phenyl-3,4-dihydro-2H-benzo[f]chromen-3-amine derivatives as potent DPP-4 inhibitors are rationally designed utilizing highly efficient 3D molecular similarity based scaffold hopping as well as electrostatic complementary methods. Those ingenious drug design strategies bring us approximate 7400-fold boost in potency. Compounds 22a and 24a are the most potent ones (IC50 ≈ 2.0 nM) with good pharmacokinetic profiles. Compound 22a demonstrated stable pharmacological effect. A 3 mg/kg oral dose provided >80% inhibition of DPP-4 activity within 24 h, which is comparable to the performance of the long-acting control omarigliptin. Moreover, the efficacy of 22a in improving the glucose tolerance is also comparable with omarigliptin. In this study, not only promising DPP-4 inhibitors as long acting antidiabetic that are clinically on demand are identified, but the target fish docking and medicinal chemistry strategies were successfully implemented.

Design, synthesis and biological evaluation of bisthiazole-based trifluoromethyl ketone derivatives as potent HDAC inhibitors with improved cellular efficacy.

Histone deacetylases (HDACs) are a class of epigenetic modulators with complex functions in histone post-translational modifications and are well known targets for antineoplastic drugs. We have previously developed a series of bisthiazole-based hydroxamic acids as novel potent HDAC inhibitors. In the present work, a new series of bisthiazole-based compounds with different zinc binding groups (ZBGs) have been designed and synthesized. Among them is compound 7, containing a trifluoromethyl ketone as the ZBG, which displays potent inhibitory activity towards human HDACs and improved antiproliferative activity in several cancer cell lines.