Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PL^Pro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PL^Pro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PL^Pro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PL^Pro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC₅₀ value below 40 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC₅₀ value below 10 μM. Among four inhibitors with an IC₅₀ value below 10 μM, SJB2-043 is the most unique in that it does not fully inhibit PL^Pro but has a noteworthy IC₅₀ value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PL^Pro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PL^Pro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.     

Discovery of a Highly Selective PLD2 Inhibitor (ML395): A New Probe with Improved Physiochemical Properties and Broad‐Spectrum Antiviral Activity against Influenza Strains

Further chemical optimization of the halopemide‐derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80‐fold PLD2 selective allosteric inhibitor (cellular PLD1, IC₅₀>30 000 nM ; cellular PLD2, IC₅₀=360 nM ). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).     

Exploration of a Library of 3,4‐(Methylenedioxy)aniline‐Derived Semicarbazones as Dual Inhibitors of Monoamine Oxidase and Acetylcholinesterase: Design,Synthesis, and Evaluation

A library of 3,4‐(methylenedioxy)aniline‐derived semicarbazones was designed, synthesized, and evaluated as monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitors for the treatment of neurodegenerative diseases. Most of the new compounds selectively inhibited MAO‐B and AChE, with IC₅₀ values in the micro‐ or nanomolar ranges. Compound 16 , 1‐(2,6‐dichlorobenzylidene)‐4‐(benzo[1,3]dioxol‐5‐yl)semicarbazide presented a balanced multifunctional profile of MAO‐A (IC₅₀=4.52±0.032 μm ), MAO‐B (IC₅₀=0.059±0.002 μm ), and AChE (IC₅₀=0.0087±0.0002 μm ) inhibition without neurotoxicity. Kinetic studies revealed that compound 16 exhibits competitive and reversible inhibition against MAO‐A and MAO‐B, and mixed‐type inhibition against AChE. Molecular docking studies further revealed insight into the possible interactions within the enzyme–inhibitor complexes. The most active compounds were found to interact with the enzymes through hydrogen bonding and hydrophobic interactions. Additionally, in silico molecular properties and ADME properties of the synthesized compounds were calculated to explore their drug‐like characteristics.     

Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (<Emphasis Type="Italic">Glycine tomentella</Emphasis>)

I-Tiao-Gung, Glycine tomentella, has been used extensively as a traditional herbal medicine to relieve physical pain, but its bioactivity has not been studied systematically. Ninety-five percent ethanol extracts of G. tomentella (GT-E) showed antioxidant activity in human plasma by prolonging the lag phase (+T_lag) of Cu²⁺-induced, LDL oxidation and were dose dependent. The+T_lag of LDL combined with 3.2 μg/mL GT-E was similar to that with 2.0 μM (ca. 0.5 μg/mL) Trolox. A similar inhibitory effect was found toward tilapia plasma LDL. In addition, GT-E inhibited tilapia thrombocyte (nucleated platelet), 5-, 12-, and 15-lipoxygenase (LOX). The IC₅₀ values were 0.43, 0.72, and 0.42 μg/mL, respectively, whereas the IC₅₀ values for nordihydroguaiaretic acid (NDGA) on 5-, 12-, and 15-LOX were 2.3, 1.6, and 1.7 μg/mL, respectively. The IC₅₀ value for cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL, whereas the IC₅₀ value by indomethacin as a positive control was 0.61 μg/mL. The prevention of LDL oxidation and the dual inhibition of LOX and COX-2 are indicative of the possible roles of I-Tiao-Gung in antiatherosclerosis and anti-inflammation.     

Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB

The novel fatty acids (2R,5Z,9Z)‐2‐methoxy‐25‐methyl‐5,9‐hexacosadienoic acid ( 1a ) and (2R,5Z,9Z)‐2‐methoxy‐24‐methyl‐5,9‐hexacosadienoic acid ( 1b ) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography‐mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP‐1 and HepG2 cells with EC_50's between 41 and 35 μg/mL. Apoptosis was not the preferred mode of cell death induced by 1a – 1b in the THP‐1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC₅₀ = 1.8 μg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC₅₀ = 0.17 mg/mL) and free living promastigotes (IC₅₀ = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC₅₀ = 5.1 μg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.     

Comparison of the effect of six compactin-related compounds on cholesterol synthesis in five human cell types

We have investigated the effect of six compactin-related compounds—mevinolin, compactin, ML-236A, monacolin X, monacolin L and dihydromonacolin L—on cholesterol synthesis in human umbilical vein endothelial cells, human small intestine epithelial cells, human hepatoma cell line HEP G2, normal human skin fibroblasts and in skin fibroblasts from a patient with familial homozygous hypercholesterolemia. The inhibition of cholesterol synthesis was found to depend on both the cell type and the type of compound used. The most effective compounds were mevinolin and compactin. Monacolin X, monacolin L and ML-236A were less effective, and dihydromonacolin L was the least efficacious. Endothelial and epithelial cells were sensitive to very low concentrations of inhibitors (IC₅₀=1.0–30 pg/mL), HEP G2 cells required higher concentrations (IC₅₀=0.01–66 ng/mL) and fibroblasts needed even higher concentrations (IC₅₀=0.1–200 ng/mL). Lactone and acid forms of the inhibitors were equally active. None of the inhibitors had any effect on either protein or fatty acid synthesis in any of the cell types studied. It can be concluded that different compactin-related compounds show a range of potencies as cholesterol synthesis inhibitors and a dose-dependent tissue-selectivity.     

Carborane-Containing Matrix Metalloprotease (MMP) Ligands as Candidates for Boron Neutron-Capture Therapy (BNCT)

Based on the previously reported potent and selective sulfone hydroxamate inhibitors SC-76276, SC-78080 (SD-2590), and SC-77964, potent MMP inhibitors have been designed and synthesized to append a boron-rich carborane cluster by employing click chemistry to target tumor cells that are known to upregulate gelatinases. Docking against MMP-2 suggests binding involving the hydroxamate zinc-binding group, key H-bonds by the sulfone moiety with the peptide backbone residues Leu82 and Leu83, and a hydrophobic interaction with the deep P1’ pocket. The more potent of the two triazole regioisomers exhibits an IC₅₀ of 3.7 nM versus MMP-2 and IC₅₀ of 46 nM versus MMP-9.     

Identification of Novel Small Molecules as Inhibitors of Hepatitis C Virus by Structure-Based Virtual Screening

Hepatitis C virus (HCV) NS3/NS4A serine protease is essential for viral replication, which is regarded as a promising drug target for developing direct-acting anti-HCV agents. In this study, sixteen novel compounds with cell-based HCV replicon activity ranging from 3.0 to 28.2 μM (IC₅₀) were successfully identified by means of structure-based virtual screening. Compound 5 and compound 11, with an IC₅₀ of 3.0 μM and 5.1 μM, respectively, are the two most potent molecules with low cytotoxicity.     

Design,Synthesis, and Biological Evaluation of New Urushiol Derivatives as Potent Class I Histone Deacetylase Inhibitors

In the present study, a novel series of 11 urushiol-based hydroxamic acid histone deacetylase (HDAC) inhibitors was designed, synthesized, and biologically evaluated. Compounds 1 – 11 exhibited good to excellent inhibitory activities against HDAC1/2/3 (IC₅₀: 42.09–240.17 nM) and HDAC8 (IC₅₀: 16.11–41.15 nM) in vitro, with negligible activity against HDAC6 (>1409.59 nM). Considering HDAC8, docking experiments revealed some important features contributing to inhibitory activity. According to Western blot analysis, select compounds could notably enhance the acetylation of histone H3 and SMC3 but not-tubulin, indicating their privileged structure is appropriate for targeting class I HDACs. Furthermore, antiproliferation assays revealed that six compounds exerted greater in vitro antiproliferative activity against four human cancer cell lines (A2780, HT-29, MDA-MB-231, and HepG2, with IC₅₀ values ranging from 2.31–5.13 μM) than suberoylanilide hydroxamic acid; administration of these compounds induced marked apoptosis in MDA-MB-231 cells, with cell cycle arrest in the G2/M phase. Collectively, specific synthesized compounds could be further optimized and biologically explored as antitumor agents.     

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC₅₀ 7.4 ± 0.7 μM) and colon cancer HT29 (IC₅₀ 9.0 ± 0.4 μM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC₅₀ 6.0 ± 1.1 μM; HT29: IC₅₀ 7.4 ± 0.6 μM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC₅₀ 7.0 ± 0.3 μM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.     

Structure‐Based Design of New KSP‐Eg5 Inhibitors Assisted by a Targeted Multicomponent Reaction

An integrated multidisciplinary approach that combined structure‐based drug design, multicomponent reaction synthetic approaches and functional characterization in enzymatic and cell assays led to the discovery of new kinesin spindle protein (KSP) inhibitors with antiproliferative activity. A focused library of new benzimidazoles obtained by a Ugi+Boc removal/cyclization reaction sequence generated low‐micromolar‐range KSP inhibitors as promising anticancer prototypes. The design and functional studies of the new chemotypes were assessed by computational modeling and molecular biology techniques. The most active compounds— 20 (IC₅₀=1.49 μM , EC₅₀=3.63 μM ) and 22 (IC₅₀=1.37 μM , EC₅₀=6.90 μM )—were synthesized with high efficiency by taking advantage of the multicomponent reactions.     

Thiazolopyrimidine Inhibitors of 2‐Methylerythritol 2,4‐Cyclodiphosphate Synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum

A library of 40 000 compounds was screened for inhibitors of 2‐methylerythritol 2,4‐cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high‐throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC₅₀ values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC₅₀ values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.     

MAO Inhibitory Activity of 2‐Arylbenzofurans versus 3‐Arylcoumarins: Synthesis,in vitro Study,and Docking Calculations

Monoamine oxidase (MAO) is an important drug target for the treatment of neurological disorders. Several 3‐arylcoumarin derivatives were previously described as interesting selective MAO‐B inhibitors. Preserving the trans‐stilbene structure, a series of 2‐arylbenzofuran and corresponding 3‐arylcoumarin derivatives were synthesized and evaluated as inhibitors of both MAO isoforms, MAO‐A and MAO‐B. In general, both types of derivatives were found to be selective MAO‐B inhibitors, with IC₅₀ values in the nano‐ to micromolar range. 5‐Nitro‐2‐(4‐methoxyphenyl)benzofuran ( 8 ) is the most active compound of the benzofuran series, presenting MAO‐B selectivity and reversible inhibition (IC₅₀=140 nM ). 3‐(4′‐Methoxyphenyl)‐6‐nitrocoumarin ( 15 ), with the same substitution pattern as that of compound 8 , was found to be the most active MAO‐B inhibitor of the coumarin series (IC₅₀=3 nM ). However, 3‐phenylcoumarin 14 showed activity in the same range (IC₅₀=6 nM ), is reversible, and also severalfold more selective than compound 15 . Docking experiments for the most active compounds into the MAO‐B and MAO‐A binding pockets highlighted different interactions between the derivative classes (2‐arylbenzofurans and 3‐arylcoumarins), and provided new information about the enzyme–inhibitor interaction and the potential therapeutic application of these scaffolds.     

Discovery of Akt Kinase Inhibitors through Structure-Based Virtual Screening and Their Evaluation as Potential Anticancer Agents

Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC₅₀ values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC₅₀ for HEK-293/IC₅₀ for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.     

Bicyclic Derivatives of the Potent Dual Aromatase–Steroid Sulfatase Inhibitor 2‐Bromo‐4‐{[(4‐cyanophenyl)(4H‐1,2,4‐triazol‐4‐yl)amino]methyl}phenylsulfamate: Synthesis,SAR, Crystal Structure,and in vitro and in vivo Activities

The design and synthesis of a series of bicyclic ring containing dual aromatase–sulfatase inhibitors (DASIs) based on the aromatase inhibitor (AI) 4‐[(4‐bromobenzyl)(4H‐1,2,4‐triazol‐4‐yl)amino]benzonitrile are reported. Biological evaluation with JEG‐3 cells revealed structure–activity relationships. The X‐ray crystal structure of sulfamate 23 was determined, and selected compounds were docked into the aromatase and steroid sulfatase (STS) crystal structures. In the sulfamate‐containing series, compounds containing a naphthalene ring are both the most potent AI ( 39 , IC_{50 AROM}=0.25 nM ) and the best STS inhibitor ( 31 , IC_{50 STS}=26 nM ). The most promising DASI is 39 (IC_{50 AROM}=0.25 nM , IC_{50 STS}=205 nM ), and this was evaluated orally in vivo at 10 mg kg^?1, showing potent inhibition of aromatase (93 %) and STS (93 %) after 3 h. Potent aromatase and STS inhibition can thus be achieved with a DASI containing a bicyclic ring system; development of such a DASI could provide an attractive new option for the treatment of hormone‐dependent breast cancer.     

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non‐cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell‐based assays. The 8‐aza‐7‐deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC₅₀=16 nm ) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell‐free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC₅₀ values ranging from 0.5 to 21 nm ). Moreover, 7‐halo‐7‐deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC₅₀ values ranging from 4.1 to 5.6 μm in HEK293 cell‐based assays.     

Tautomycetin Synthetic Analogues: Selective Inhibitors of Protein Phosphatase I

Ser/Thr protein phosphatases (PPs) regulate a substantial range of cellular processes with protein phosphatases 1 (PP1) and 2 A (PP2A) accounting for over 90 % of the activity within cells. Nevertheless, tools to study PPs are limited as PPs inhibitors, particularly those selective for PP1 inhibition, are relatively scarce. Two examples of PP1-selective inhibitors, which share structural similarities, are tautomycin (TTM) and tautomycetin (TTN). This work describes the development of PP1/PP2A inhibitors that incorporate key structural features of TTM and TTN and are designed to conserve regions known to bind the active site of PP1/PP2A but vary regions that differentially contact the hydrophobic groove of PP1/PP2A. In all 28 TTN analogues were synthetically generated that inhibit PP1/PP2A activity at <250 mM; seven possessed inhibition activity at 100 nM. The IC₅₀ values were determined for the seven most active analogues, which ranged from 34 to 1500 nM (PP1) and 70 to 6800 nM (PP2A). Four of the seven analogues possessed PP1 selectivity, and one demonstrated eightfold selectivity in the nanomolar range (PP1 IC₅₀=34 nM, PP2A IC₅₀=270 nM). A rationale is given for the observed differences in selectivity.     

Synthesis,Biological Evaluation and Molecular Modeling Studies of Propargyl‐Containing 2,4,6‐Trisubstituted Pyrimidine Derivatives as Potential Anti‐Parkinson Agents

Monoamine oxidase B (MAO‐B) inhibitors are potential drug candidates for the treatment of various neurological disorders including Parkinson's disease. A total of 20 new propargyl‐containing 2,4,6‐trisubstituted pyrimidine derivatives were synthesized and screened for MAO inhibition using Amplex Red assays. All the synthesized compounds were found to be reversible and selective inhibitors of the MAO‐B isoform at sub‐micromolar concentrations. MVB3 was the most potent MAO‐B inhibitor with an IC₅₀ value of 0.38±0.02 μμ , whereas MVB6 (IC₅₀=0.51±0.04 μμ ) and MVB16 (IC₅₀=0.48±0.06 μμ ) were the most selective for MAO‐B with a selectivity index of more than 100‐fold. In cytotoxic studies, these compounds were found to be nontoxic to human neuroblastoma SH‐SY5Y cells at concentrations of 25 μm . MVB6 was found to decrease the intracellular level of reactive oxygen species to 68 % at 10 μm concentration, whereas other compounds did not produce significant changes in reactive oxygen species levels. In molecular modeling studies, MVB3 displayed strong binding affinity for the MAO‐B isoform with a dock score of ?10.45, in agreement with the observed activity. All the compounds fitted well in the hydrophobic cavity of MAO‐B. Thus, propargyl‐substituted pyrimidine derivatives can be promising leads in the development of potent, selective and reversible MAO‐B inhibitors for the treatment of Parkinson's disease.     

Asymmetric Synthesis and Biological Evaluation of Glycosidic Prodrugs for a Selective Cancer Therapy

A severe limitation in cancer therapy is the often insufficient differentiation between malign and benign tissue using known chemotherapeutics. One approach to decrease side effects is antibody‐directed enzyme prodrug therapy (ADEPT). We have developed new glycosidic prodrugs such as (?)‐(1S)‐ 26 b based on the antibiotic (+)‐duocarmycin SA ((+)‐ 1 ) with a QIC₅₀ value of 3500 (QIC₅₀=IC₅₀ of prodrug/IC₅₀ of prodrug+enzyme) and an IC₅₀ value for the corresponding drug (prodrug+enzyme) of 16 pM . The asymmetric synthesis of the precursor (?)‐(1S)‐ 19 was performed by arylation of the enantiomerically pure epoxide (+)‐(S)‐ 29 (≥98 % ee).     

Synthesis,In Vitro and In Silico Anticancer Activity of New 4-Methylbenzamide Derivatives Containing 2,6-Substituted Purines as Potential Protein Kinases Inhibitors

A novel class of potential protein kinase inhibitors 7–16 was synthesized in high yields using various substituted purines. The most promising compounds, 7 and 10, exhibited inhibitory activity against seven cancer cell lines. The IC₅₀ values for compounds 7 and 10 were 2.27 and 2.53 μM for K562 cells, 1.42 and 1.52 μM for HL-60 cells, and 4.56 and 24.77 μM for OKP-GS cells, respectively. In addition, compounds 7 and 10 dose-dependently induced the apoptosis and cell cycle arrest at G2/M phase, preventing the cell division of OKP-GS cells. Compounds 7, 9, and 10 showed 36–45% inhibitory activity against PDGFRα and PDGFRβ at the concentration of 1 μM. Molecular modeling experiments showed that obtained compounds could bind to PDGFRα as either type 1 (compound 7, ATP-competitive) or type 2 (compound 10, allosteric) inhibitors, depending on the substituent in the amide part of the molecule.