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).

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.

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.

Curcusone D, a novel ubiquitin-proteasome pathway inhibitor via ROS-induced DUB inhibition, is synergistic with bortezomib against multiple myeloma cell growth.

BACKGROUND: Ubiquitin-proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM). METHODS: Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H2DCFDA. RESULTS: Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells. CONCLUSIONS: Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib. GENERAL SIGNIFICANCE: The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.

Design, synthesis and biological evaluation of isoquinoline-based derivatives as novel histone deacetylase inhibitors.

The design, synthesis and biological evaluation of a series of isoquinoline-based hydroxamic acid compounds as novel HDACs inhibitors were reported herein. A detailed SAR study showed most of the compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC50 values of compound 10 c against HDAC1, 3, 6 were 4.17 ± 0.11 nM, 4.00 ± 0.10 nM, 3.77 ± 0.07 nM, respectively. Most of the compounds showed great anti-proliferative activities against RPMI 8226, HCT 116 and Hep G2 cells. The IC50 values of compounds 10 a-h against RPMI 8226 cancer cell proliferation were all below 1 µM. HCT 116 cell was sensitive to the compounds 10 a, 10 f-g and 18 a with the IC50 values <0.3 µM. The active compounds 10a-d did not show inhibitory activity against hERG channel. All these evidence indicated these compounds had great potential as HDACs inhibitors for the further development.

Design, synthesis and biological evaluation of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel histone deacetylase inhibitors.

A series of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel HDACs inhibitors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC50 values of compound 10r against HDAC1, HDAC3, HDAC6 was 1.14 ± 0.03 nM, 3.56 ± 0.08 nM, 11.43 ± 0.12 nM. Compound 10r noticeably up-regulated the level of histone H3 acetylation compared to the SAHA. Most of the compounds showed the strong anti-proliferative activity against human cancer cell lines including RMPI8226 and HCT-116. The IC50 values of Compounds 10r and 10t against RPMI8226 was 2.39 ± 0.20 µM, 1.41 ± 0.44 µM, respectively, and the HCT-116 was sensitive to the compounds 10h, 10 m, 10r, 10 w with the IC50 values <1.9 µM.

(R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives as potent inhibitors of dipeptidyl peptidase-4: design, synthesis, biological evaluation, and molecular modeling.

A series of (R)-3-amino-1-((3aS,7aS)-octahydro-1H-indol-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one derivatives was designed, synthesized, and evaluated as novel inhibitors of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes. Most of the synthesized compounds demonstrated good inhibition activities against DPP-4. Among these, compounds 3e, 4c, 4l, and 4n exhibited prominent inhibition activities against DPP-4, with IC50s of 0.07, 0.07, 0.14, and 0.17µM, respectively. The possible binding modes of compounds 3e and 4n with dipeptidyl peptidase-4 were also explored by molecular docking simulation. These potent DPP-4 inhibitors were optimized for the absorption, distribution, metabolism, and excretion (ADME) properties, and compound 4n displayed an attractive pharmacokinetic profile (F=96.3%, t1/2=10.5h).

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.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.

Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees.

Worker and queen bees are genetically indistinguishable. However, queen bees are fertile, larger and have a longer lifespan than their female worker counterparts. Differential feeding of larvae with royal jelly controls this caste switching. There is emerging evidence that the queen-bee phenotype is driven by epigenetic mechanisms. In this study, we show that royal jelly--the secretion produced by the hypopharyngeal and mandibular glands of worker bees--has histone deacetylase inhibitor (HDACi) activity. A fatty acid, (E)-10-hydroxy-2-decenoic acid (10HDA), which accounts for up to 5% of royal jelly, harbours this HDACi activity. Furthermore, 10HDA can reactivate the expression of epigenetically silenced genes in mammalian cells. Thus, the epigenetic regulation of queen-bee development is probably driven, in part, by HDACi activity in royal jelly.

The discovery of colchicine-SAHA hybrids as a new class of antitumor agents.

A novel class of colchicine-SAHA hybrids were designed and synthesised based on the synergistic antitumor effect of tubulin inhibitors and histone deacetylases (HDAC) inhibitors. To the best of our knowledge, this is the first design of molecules that are dual inhibitors of tubulin and HDAC. Biological evaluations of these compounds included the inhibitory activity of HDAC, in vitro cell cycle analysis in BEL-7402 cells as well as cytotoxicity in five cancer cell lines.

The discovery and optimization of novel dual inhibitors of topoisomerase II and histone deacetylase.

A novel class of podophyllotoxin derivatives have been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities towards histone deacetylases and Topo II and their cytotoxicities in cancer cell lines were evaluated. The aromatic capping group connection, linker length and zinc-binding group were systematically varied and preliminary conclusions regarding structure-activity relationships are discussed. Among all of the synthesized hybrid compounds, compound 24 d showed the most potent HDAC inhibitory activity at a low nanomolar level and exhibited powerful antiproliferative activity towards HCT116 colon carcinoma cells at a low micromolar level. Further exploration of this series led to the discovery of potent dual inhibitor 32, which exhibited the strongest in vitro cytotoxic activity.

The design and synthesis of a new class of RTK/HDAC dual-targeted inhibitors.

Over the years, the development of targeted medicines has made significant achievements. As a typical example, receptor tyrosine kinases (RTK) inhibitors have become important chemotherapy drugs for a variety of cancers. However, the effectiveness of these agents is always hindered by poor response rates and acquired drug resistance. In order to overcome these limitations, several dual-targeted inhibitors with quinazoline core were designed and synthesized. Though these compounds can simultaneously inhibit histone deacetylases (HDAC) as well as RTK, the structure-activity relationship (SAR) is still not clear enough. To further explore this type of dual-targeted inhibitors, a new class of quinazoline derivatives were designed and synthesized. Their activity evaluations include in vitro inhibitory activity of HDAC, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). The SAR study indicated that the introduction of polar group such as hydroxamate on the 4-position of the quinazoline core is more likely to provide a potent HDACi/HER2i hybrid rather than HDACi/EGFRi molecule.

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.

CCLab--a multi-objective genetic algorithm based combinatorial library design software and an application for histone deacetylase inhibitor design.

The introduction of the multi-objective optimization has dramatically changed the virtual combinatorial library design, which can consider many objectives simultaneously, such as synthesis cost and drug-likeness, thus may increase positive rates of biological active compounds. Here we described a software called CCLab (Combinatorial Chemistry Laboratory) for combinatorial library design based on the multi-objective genetic algorithm. Tests of the convergence ability and the ratio to re-take the building blocks in the reference library were conducted to assess the software in silico, and then it was applied to a real case of designing a 5×6 HDAC inhibitor library. Sixteen compounds in the resulted library were synthesized, and the histone deactetylase (HDAC) enzymatic assays proved that 14 compounds showed inhibitory ratios more than 50% against tested 3 HDAC enzymes at concentration of 20 µg/mL, with IC(50) values of 3 compounds comparable to SAHA. These results demonstrated that the CCLab software could enhance the hit rates of the designed library and would be beneficial for medicinal chemists to design focused library in drug development (the software can be downloaded at: http://202.127.30.184:8080/drugdesign.html).

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.

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).

Synthesis and biological evaluation of piperamide analogues as HDAC inhibitors.

Two natural piperamides (piperlonguminine and refrofractamide A) and their derivatives were synthesized and evaluated for inhibitory activity against histone deacetylases, as well as the HCT-116 human colon cancer cell line. The preliminary structure activity relationship was discussed. Compounds featuring a hydroxamic acid moiety exhibited moderate HDAC activity and in vitro cytotoxicity.