Publications by authors named "Arun K Ghosh"

336 Publications

Novel HIV PR inhibitors with C4-substituted bis-THF and bis-fluoro-benzyl target the two active site mutations of highly drug resistant mutant PR.

Biochem Biophys Res Commun 2021 Jun 7;566:30-35. Epub 2021 Jun 7.

Department of Biology, Georgia State University, Atlanta, GA, 30303, USA; Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA. Electronic address:

The emergence of multidrug resistant (MDR) HIV strains severely reduces the effectiveness of antiretroviral therapy. Clinical inhibitor darunavir (1) has picomolar binding affinity for HIV-1 protease (PR), however, drug resistant variants like PR show poor inhibition by 1, despite the presence of only two mutated residues in the inhibitor-binding site. Antiviral inhibitors that target MDR proteases like PR would be valuable as therapeutic agents. Inhibitors 2 and 3 derived from 1 through substitutions at P1, P2 and P2' positions exhibit 3.4- to 500-fold better inhibition than clinical inhibitors for PR with the exception of amprenavir. Crystal structures of PR/2 and PR/3 reveal how these inhibitors target the two active site mutations of PR. The substituted methoxy P2 group of 2 forms new interactions with G48V mutation, while the modified bis-fluoro-benzyl P1 group of 3 forms a halogen interaction with V82S mutation, contributing to improved inhibition of PR.
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http://dx.doi.org/10.1016/j.bbrc.2021.05.094DOI Listing
June 2021

Highly Diastereoselective Intramolecular Asymmetric Oxidopyrylium-olefin [5 + 2] Cycloaddition and Synthesis of 8-Oxabicyclo[3.2.1]oct-3-enone Containing Ring Systems.

J Org Chem 2021 May 20. Epub 2021 May 20.

Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States.

We have the investigated base mediated asymmetric intramolecular oxidopyrylium-alkene [5 + 2]-cycloaddition reaction which resulted in the synthesis of functionalized tricyclic ring systems containing an 8-oxabicyclo[3.2.1]octane core. Intramolecular cycloaddition constructed two new rings, three new stereogenic centers, and provided a tricyclic cycloadduct with high diastereoselectivity and isolated yield. We incorporated an α-chiral center and an alkoxy alkene tether on the substrates and examined the effect of the size of alkyl groups and alkene tether length on diastereoselectivity. The requisite substrates for the oxidopyrylium-alkene cycloaddition reaction were synthesized in a few steps involving alkylation of optically active α-hydroxy amide, furyllithium addition, reduction of resulting ketone, and Achmatowicz reaction followed by acylation of a lactol intermediate. We have proposed stereochemical models for the [5 + 2] cycloaddition reaction via the oxidopyrylium ylide. Interestingly, the alkoxy substituent on the stereocenter and the chain length are responsible for the degree of stereoselectivity of the cycloadduct.
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http://dx.doi.org/10.1021/acs.joc.1c00600DOI Listing
May 2021

Spliceostatins and Derivatives: Chemical Syntheses and Biological Properties of Potent Splicing Inhibitors.

J Nat Prod 2021 05 11;84(5):1681-1706. Epub 2021 May 11.

Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, United States.

Spliceostatins and thailanstatins are intriguing natural products due to their structural features as well as their biological significance. This family of natural products has been the subject of immense synthetic interest because they exhibit very potent cytotoxicity in representative human cancer cell lines. The cytotoxic properties of these natural products are related to their ability to inhibit spliceosomes. FR901564 and spliceostatins have been shown to inhibit spliceosomes by binding to their SF3B component. Structurally, these natural products contain two highly functionalized tetrahydropyran rings with multiple stereogenic centers joined by a diene moiety and an acyclic side chain linked with an amide bond. Total syntheses of this family of natural products led to the development of useful synthetic strategies, which enabled the synthesis of potent derivatives. The spliceosome modulating properties of spliceostatins and synthetic derivatives opened the door for understanding the underlying spliceosome mechanism as well as the development of new therapies based upon small-molecule splicing modulators. This review outlines the total synthesis of spliceostatins, synthetic studies of structural derivatives, and their bioactivity.
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http://dx.doi.org/10.1021/acs.jnatprod.1c00100DOI Listing
May 2021

Enantioselective Total Synthesis of (+)-EBC-23, a Potent Anticancer Agent from the Australian Rainforest.

J Org Chem 2021 May 19;86(9):6351-6360. Epub 2021 Apr 19.

We describe here an enantioselective synthesis of (+)-EBC-23, a potent anticancer agent from the Australian rainforest. Our convergent synthesis features a [3+2] dipolar cycloaddition of an olefin-bearing 1,3- diol unit and an oxime segment containing 1,2- diol functionality as the key step. The segments were synthesized in a highly enantioselective manner using Noyori asymmetric hydrogenation of a β-keto ester and Sharpless asymmetric dihydroxylation of an α,β-unsaturated ester. Cycloaddition provided isoxazoline derivative which upon hydrogenolysis furnished the β-hydroxy ketone expediently. A one-pot, acid-catalyzed reaction removed the isopropylidene group, promoted spirocyclization, constructed the complex spiroketal lactone core, and furnished EBC-23 and its C11 epimer. The C11 epimer was also converted to EBC-23 by chemoselective oxidation and reduction sequence. The present synthesis provides convenient access to this family of natural products in an efficient manner.
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http://dx.doi.org/10.1021/acs.joc.1c00172DOI Listing
May 2021

Herboxidiene Features That Mediate Conformation-Dependent SF3B1 Interactions to Inhibit Splicing.

ACS Chem Biol 2021 03 22;16(3):520-528. Epub 2021 Feb 22.

Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Employing an inactive herboxidiene analog (iHB) as a competitor, we investigated factors that influence inhibitor interactions with SF3B to interfere with pre-mRNA splicing . Order-of-addition experiments show that inhibitor interactions are long lasting and affected by both temperature and the presence of ATP. Our data are also consistent with the model that not all SF3B conformations observed in structural studies are conducive to productive inhibitor interactions. Notably, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure-activity relationship analysis of the splicing inhibitor herboxidiene. We identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In the context of recent structures of SF3B bound to inhibitor, our results lead us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 side chains in the inhibitor binding channel are needed to maintain inhibitor occupancy while counteracting the SF3B transition to a closed state that is required for stable U2 snRNP interactions with the intron.
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http://dx.doi.org/10.1021/acschembio.0c00965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189251PMC
March 2021

A Structure-Based Discovery Platform for BACE2 and the Development of Selective BACE Inhibitors.

ACS Chem Neurosci 2021 02 5;12(4):581-588. Epub 2021 Feb 5.

The ability to perform routine structure-guided drug design for selective BACE inhibitors has been limited because of the lack of robust platform for BACE2 expression, purification, and crystallization. To overcome this limitation, we developed a platform that produces 2-3 mg of pure BACE2 protein per liter of culture, and we used this protein to design macrocyclic compounds that potently and selectively inhibit BACE1 over BACE2. Compound was found to potently inhibit BACE 1 ( = 5 nM) with a selectivity of 214-fold over BACE2. The X-ray crystal structures of unbound BACE2 (2.2 Å) and BACE2 bound to compound (3.0 Å and = 7 nM) were determined and compared to the X-ray structures of BACE1 revealing the S1-S3 subsite as a selectivity determinant. This platform should enable a more rapid development of new and selective BACE inhibitors for the treatment of Alzheimer's disease or type II diabetes.
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http://dx.doi.org/10.1021/acschemneuro.0c00629DOI Listing
February 2021

A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication.

Nat Commun 2021 01 28;12(1):668. Epub 2021 Jan 28.

Department of Refractory Viral Infections, National Center for Global Health and Medicine Research Institute, Tokyo, Japan.

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (M). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC values of 15 ± 4 and 4.2 ± 0.7 μM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with M and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead M inhibitor for the development of therapeutics for SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41467-021-20900-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843602PMC
January 2021

Design and synthesis of herboxidiene derivatives that potently inhibit splicing.

Org Biomol Chem 2021 02;19(6):1365-1377

Department of Molecular Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.

Herboxidiene is a potent antitumor agent that targets the SF3B subunit of the spliceosome. Herboxidiene possesses a complex structural architecture with nine stereocenters and design of potent less complex structures would be of interest as a drug lead as well as a tool for studying SF3B1 function in splicing. We investigated a number of C-6 modified herboxidiene derivatives in an effort to eliminate this stereocenter and, also to understand the importance of this functionality. The syntheses of structural variants involved a Suzuki-Miyaura cross-coupling reaction as the key step. The functionalized tetrahydrofuran core has been constructed from commercially available optically active tri-O-acetyl-d-glucal. We investigated the effect of these derivatives on splicing chemistry. The C-6 alkene derivative showed very potent splicing inhibitory activity similar to herboxidiene. Furthermore, the C-6 gem-dimethyl derivative also exhibited very potent in vitro splicing inhibitory activity comparable to herboxidiene.
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http://dx.doi.org/10.1039/d0ob02532aDOI Listing
February 2021

Synthesis of amide derivatives for electron deficient amines and functionalized carboxylic acids using EDC and DMAP and a catalytic amount of HOBt as the coupling reagents.

Tetrahedron Lett 2021 Jan 25;63. Epub 2020 Dec 25.

Department of Chemistry, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, United States.

A convenient protocol for amide bond formation for electron deficient amines and carboxylic acids is described. Amide coupling of aniline derivatives has been investigated with a number of reagents under a variety of reaction conditions. The use of 1 equivalent of EDC and 1 equivalent of DMAP, catalytic amount of HOBt and DIPEA provided the best results. This method is amenable to the synthesis of a range of functionalized amide derivatives with electron deficient and unreactive amines.
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http://dx.doi.org/10.1016/j.tetlet.2020.152719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808253PMC
January 2021

The Chiron Approach to (3,3,6)-Hexahydrofuro[2,3-]furan-3-ol, a Key Subunit of HIV-1 Protease Inhibitor Drug, Darunavir.

J Org Chem 2021 01 3;86(1):1216-1222. Epub 2020 Dec 3.

Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States.

We describe an enantioselective synthesis of (3,3,6)-hexahydrofuro[2,3-]furan-3-ol which is a key subunit of darunavir, a widely used HIV-1 protease inhibitor drug for the treatment of HIV/AIDS patients. The synthesis was achieved in optically pure form utilizing commercially available sugar derivatives as the starting material. The key steps involve a highly stereoselective substrate-controlled hydrogenation, a Lewis acid catalyzed anomeric reduction of a 1,2--isopropylidene-protected glycofuranoside, and a Baeyer-Villiger oxidation of a tetrahydrofuranyl-2-aldehyde derivative. This optically active ligand alcohol was converted to darunavir efficiently.
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http://dx.doi.org/10.1021/acs.joc.0c02396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894212PMC
January 2021

Design, Synthesis, and X-ray Studies of Potent HIV-1 Protease Inhibitors with P2-Carboxamide Functionalities.

ACS Med Chem Lett 2020 Oct 3;11(10):1965-1972. Epub 2020 Mar 3.

Department of Refractory Viral Infections, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan.

The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with ()-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2'-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor -bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549267PMC
October 2020

GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection.

mBio 2020 08 20;11(4). Epub 2020 Aug 20.

Department of Refractory Viral Infections, National Center for Global Health and Medicine Research Institute, Tokyo, Japan

We assessed various newly generated compounds that target the main protease (M) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC] = 2.8 μM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of M, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography-mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds. Targeting the main protease (M) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of M, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.
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http://dx.doi.org/10.1128/mBio.01833-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441487PMC
August 2020

Lewis Acid-Catalyzed Vinyl Acetal Rearrangement of 4,5-Dihydro-1,3-dioxepines: Stereoselective Synthesis of and 2,3-Disubstituted Tetrahydrofurans.

J Org Chem 2020 08 5;85(16):10399-10412. Epub 2020 Aug 5.

Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States.

Lewis acid-catalyzed rearrangements of 4,5-dihydro-1,3-dioxepines have been investigated. Rearrangement of vinyl acetals under a variety of conditions resulted in - and 2,3-disubstituted tetrahydrofuran derivatives in a highly stereoselective manner. Rearrangements at lower temperatures typically provided the -2,3-disubstituted tetrahydrofuran carbaldehydes. At higher temperatures, the corresponding -2,3-disubstituted tetrahydrofuran carbaldehydes are formed. The requisite substrates for the vinyl acetal rearrangement were synthesized via ring-closing olefin metathesis of bis(allyoxy)methyl derivatives using Grubbs second-generation catalyst followed by olefin isomerization using a catalytic amount of RuCl(PPh). We examined the substrate scope using substituted aromatic and aliphatic derivatives. Additionally, the rearrangement was utilized in the synthesis of a stereochemically-defined bis-tetrahydrofuran (bis-THF) derivative, which is one of the key structural elements of darunavir, an FDA-approved drug for the treatment of HIV/AIDS.
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http://dx.doi.org/10.1021/acs.joc.0c00390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898272PMC
August 2020

Single atom changes in newly synthesized HIV protease inhibitors reveal structural basis for extreme affinity, high genetic barrier, and adaptation to the HIV protease plasticity.

Sci Rep 2020 06 30;10(1):10664. Epub 2020 Jun 30.

HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, MD, United States.

HIV-1 protease inhibitors (PIs), such as darunavir (DRV), are the key component of antiretroviral therapy. However, HIV-1 often acquires resistance to PIs. Here, seven novel PIs were synthesized, by introducing single atom changes such as an exchange of a sulfur to an oxygen, scission of a single bond in P2'-cyclopropylaminobenzothiazole (or -oxazole), and/or P1-benzene ring with fluorine scan of mono- or bis-fluorine atoms around DRV's scaffold. X-ray structural analyses of the PIs complexed with wild-type Protease (PR) and highly-multi-PI-resistance-associated PR revealed that the PIs better adapt to structural plasticity in PR with resistance-associated amino acid substitutions by formation of optimal sulfur bond and adaptation of cyclopropyl ring in the S2'-subsite. Furthermore, these PIs displayed increased cell permeability and extreme anti-HIV-1 potency compared to DRV. Our work provides the basis for developing novel PIs with high potency against PI-resistant HIV-1 variants with a high genetic barrier.
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http://dx.doi.org/10.1038/s41598-020-65993-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326966PMC
June 2020

Copper-Catalyzed Stille Cross-Coupling Reaction and Application in the Synthesis of the Spliceostatin Core Structure.

J Org Chem 2020 06 9;85(12):8111-8120. Epub 2020 Jun 9.

Department of Molecular Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California 95064, United States.

An efficient palladium-free Stille cross-coupling reaction of allylic bromides and functionalized organostannylfuran using catalytic copper halide has been developed. The coupling reaction was optimized using CuI and low catalyst loading (down to 5 mol %). The reaction was conveniently carried out at ambient temperature in the presence of inorganic base to afford the coupling product in good-to-excellent yields. The utility of this reaction was demonstrated in the synthesis of a furan with sensitive functionalities. A sulfolene moiety was utilized as a masking group for the sensitive diene. Noyori asymmetric reduction, Achmatowicz reaction, and Kishi reduction steps converted sulfolene to a highly substituted tetrahydropyran intermediate used in the synthesis of the highly potent antitumor agents, spliceostatins, and their derivatives.
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http://dx.doi.org/10.1021/acs.joc.0c00976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900942PMC
June 2020

Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

J Med Chem 2020 05 29;63(9):4867-4879. Epub 2020 Apr 29.

Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425579PMC
May 2020

Drug Development and Medicinal Chemistry Efforts toward SARS-Coronavirus and Covid-19 Therapeutics.

ChemMedChem 2020 06 7;15(11):907-932. Epub 2020 May 7.

Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.

The COVID-19 pandemic caused by SARS-CoV-2 infection is spreading at an alarming rate and has created an unprecedented health emergency around the globe. There is no effective vaccine or approved drug treatment against COVID-19 and other pathogenic coronaviruses. The development of antiviral agents is an urgent priority. Biochemical events critical to the coronavirus replication cycle provided a number of attractive targets for drug development. These include, spike protein for binding to host cell-surface receptors, proteolytic enzymes that are essential for processing polyproteins into mature viruses, and RNA-dependent RNA polymerase for RNA replication. There has been a lot of ground work for drug discovery and development against these targets. Also, high-throughput screening efforts have led to the identification of diverse lead structures, including natural product-derived molecules. This review highlights past and present drug discovery and medicinal-chemistry approaches against SARS-CoV, MERS-CoV and COVID-19 targets. The review hopes to stimulate further research and will be a useful guide to the development of effective therapies against COVID-19 and other pathogenic coronaviruses.
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http://dx.doi.org/10.1002/cmdc.202000223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264561PMC
June 2020

Fluorescent Probes for Monitoring Serine Ubiquitination.

Biochemistry 2020 04 27;59(13):1309-1313. Epub 2020 Mar 27.

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47906, United States.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of effectors utilize NAD to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.
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http://dx.doi.org/10.1021/acs.biochem.0c00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294441PMC
April 2020

Urea Derivatives in Modern Drug Discovery and Medicinal Chemistry.

J Med Chem 2020 03 2;63(6):2751-2788. Epub 2019 Dec 2.

Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.

The urea functionality is inherent to numerous bioactive compounds, including a variety of clinically approved therapies. Urea containing compounds are increasingly used in medicinal chemistry and drug design in order to establish key drug-target interactions and fine-tune crucial drug-like properties. In this perspective, we highlight physicochemical and conformational properties of urea derivatives. We provide outlines of traditional reagents and chemical procedures for the preparation of ureas. Also, we discuss newly developed methodologies mainly aimed at overcoming safety issues associated with traditional synthesis. Finally, we provide a broad overview of urea-based medicinally relevant compounds, ranging from approved drugs to recent medicinal chemistry developments.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266097PMC
March 2020

A novel HIV-1 protease inhibitor, GRL-044, has potent activity against various HIV-1s with an extremely high genetic barrier to the emergence of HIV-1 drug resistance.

Glob Health Med 2019 Oct;1(1):36-48

Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

We designed, synthesized, and identified two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL- 037 and GRL-044, containing P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), P1-benzene and P1-methoxybenzene, respectively, and P2'-isopropyl-aminobenzothiazole (Ip-Abt), based on the structure of the prototypic PI, darunavir (DRV). The 50% inhibitory concentrations (ICs) of GRL-037 and GRL-044 against wild-type HIV-1 were 0.042 and 0.0028-0.0033 nM with minimal cytotoxicity profiles compared to the IC values of four most potent FDA-approved PIs, ranging from 2.6 to 70 nM. GRL-044 was also potent against HIV-2EHO (IC=0.0004 nM) and various PI-resistant HIV-1 variants (IC ranging from 0.065 to 19 nM). In the selection assays we conducted, the emergence of HIV-1 variants resistant to GRL-044 was significantly delayed compared to that against DRV. Thermal stability test using differential scanning fluorimetry employing purified HIV-1 protease (PR) and SYPRO Orange showed that both GRL-037 and GRL-044 tightly bound to PR. A28S substitution emerged in the homologous recombination-based selection assays with GRL-044. Structural analyses showed that the larger size of GRL-044 over DRV, enabling GRL-044 to fit better to the hydrophobic cavity of protease, contributed to the greater potency of GRL- 044 against HIV-1. Structural analyses also suggested that the van der Waals surface contact of GRL-044 with A28' appears to be better compared to that of DRV because of the larger surface of Ip-Abt of GRL-044, which may be partially responsible for the emergence of A28S. The present antiviral data and structural features of GRL-044 should provide molecular insights for further design and development of potent and "resistance-repellant" novel PIs.
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http://dx.doi.org/10.35772/ghm.2019.01003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731189PMC
October 2019

Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors.

Biochemistry 2019 11 22;58(44):4424-4435. Epub 2019 Oct 22.

BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer's Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. We therefore developed a platform for expression and purification of BACE1, including protein refolding from inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ∼15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants () obtained from the kinetic studies are in agreement with dissociation constants () that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.
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http://dx.doi.org/10.1021/acs.biochem.9b00714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284891PMC
November 2019

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies.

ChemMedChem 2019 11 4;14(21):1863-1872. Epub 2019 Oct 4.

Departments of Hematology and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan.

We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2' ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme K but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIV and HIV viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.
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http://dx.doi.org/10.1002/cmdc.201900508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842059PMC
November 2019

Potent antiviral HIV-1 protease inhibitor combats highly drug resistant mutant PR20.

Biochem Biophys Res Commun 2019 10 29;519(1):61-66. Epub 2019 Aug 29.

Department of Biology, Georgia State University, Atlanta, GA, 30303, USA; Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA. Electronic address:

Drug-resistance threatens effective treatment of HIV/AIDS. Clinical inhibitors, including darunavir (1), are ineffective for highly resistant protease mutant PR20, however, antiviral compound 2 derived from 1 with fused tricyclic group at P2, extended amino-benzothiazole P2' ligand and two fluorine atoms on P1 shows 16-fold better inhibition of PR20 enzyme activity. Crystal structures of PR20 and wild-type PR complexes reveal how the extra groups of 2 counteract the expanded ligand-binding pocket, dynamic flaps, and faster dimer dissociation of PR20.
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http://dx.doi.org/10.1016/j.bbrc.2019.08.126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251940PMC
October 2019

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design, synthesis, and biological evaluation.

Bioorg Med Chem Lett 2019 09 5;29(18):2565-2570. Epub 2019 Aug 5.

Department of Refractory Viral Infections, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655, Japan; Experimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer Institute, Bethesda, MD 20892, USA; Division of Clinical Sciences, Kumamoto University Hospital, Kumamoto 860-8556, Japan.

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory K value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.
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http://dx.doi.org/10.1016/j.bmcl.2019.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711809PMC
September 2019

A Photochemical Route to Optically Active Hexahydro-4-furopyranol, a High-Affinity P2 Ligand for HIV-1 Protease Inhibitors.

J Org Chem 2019 08 16;84(15):9801-9805. Epub 2019 Jul 16.

Department of Chemistry and Department of Medicinal Chemistry , Purdue University , 560 Oval Drive , West Lafayette , Indiana 47907 , United States.

We describe here the syntheses of optically pure (3a,4,7a)-hexahydro-4-furo[2,3-]pyran-4-ol and (3a,4,7a)-hexahydro-4-furo[2,3-]pyran-4-ol. These stereochemically defined heterocycles are important high-affinity P2 ligands for a variety of highly potent HIV-1 protease inhibitors. The key steps involve an efficient Paternò-Büchi [2 + 2] photocycloaddition, catalytic hydrogenation, acid-catalyzed cyclization to form the racemic ligand alcohol, and an enzymatic resolution with immobilized Amano Lipase PS-30. Optically active ligands (-)- and (+)- were obtained with high enantiomeric purity. Enantiomer (-)- has been converted to potent HIV-1 protease inhibitor .
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http://dx.doi.org/10.1021/acs.joc.9b01361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048240PMC
August 2019

Structural studies of antiviral inhibitor with HIV-1 protease bearing drug resistant substitutions of V32I, I47V and V82I.

Biochem Biophys Res Commun 2019 06 12;514(3):974-978. Epub 2019 May 12.

Department of Biology, Georgia State University, Atlanta, GA, 30303, USA. Electronic address:

HIV-1 protease inhibitors are effective in HIV/AIDS therapy, although drug resistance is a severe problem. This study examines the effects of four investigational inhibitors against HIV-1 protease with drug resistant mutations of V32I, I47V and V82I (PR) that model the inhibitor-binding site of HIV-2 protease. These inhibitors contain diverse chemical modifications on the darunavir scaffold and form new interactions with wild type protease, however, the measured inhibition constants for PR mutant range from 17 to 40 nM or significantly worse than picomolar values reported for wild type enzyme. The X-ray crystal structure of PR mutant in complex with inhibitor 1 at 1.5 Å resolution shows minor changes in interactions with inhibitor compared with the corresponding wild type PR complex. Instead, the basic amine at P2 of inhibitor together with mutation V82I induces two alternate conformations for the side chain of Arg8 with new interactions with inhibitor and Leu10. Hence, inhibition is influenced by small coordinated changes in hydrophobic interactions.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601333PMC
June 2019

Novel Protease Inhibitors Containing C-5-Modified -Tetrahydrofuranylurethane and Aminobenzothiazole as P2 and P2' Ligands That Exert Potent Antiviral Activity against Highly Multidrug-Resistant HIV-1 with a High Genetic Barrier against the Emergence of Drug Resistance.

Antimicrob Agents Chemother 2019 08 25;63(8). Epub 2019 Jul 25.

Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

Combination antiretroviral therapy has achieved dramatic reductions in the mortality and morbidity in people with HIV-1 infection. Darunavir (DRV) represents a most efficacious and well-tolerated protease inhibitor (PI) with a high genetic barrier to the emergence of drug-resistant HIV-1. However, highly DRV-resistant variants have been reported in patients receiving long-term DRV-containing regimens. Here, we report three novel HIV-1 PIs (GRL-057-14, GRL-058-14, and GRL-059-14), all of which contain a P2-amino-substituted--tetrahydrofuranylurethane (-THF) and a P2'-cyclopropyl-amino-benzothiazole (Cp-Abt). These PIs not only potently inhibit the replication of wild-type HIV-1 (50% effective concentration [EC], 0.22 nM to 10.4 nM) but also inhibit multi-PI-resistant HIV-1 variants, including highly DRV-resistant HIV (EC, 1.6 nM to 30.7 nM). The emergence of HIV-1 variants resistant to the three compounds was much delayed in selection experiments compared to resistance to DRV, using a mixture of 11 highly multi-PI-resistant HIV-1 isolates as a starting HIV-1 population. GRL-057-14 showed the most potent anti-HIV-1 activity and greatest thermal stability with wild-type protease, and potently inhibited HIV-1 protease's proteolytic activity ( value, 0.10 nM) among the three PIs. Structural models indicate that the C-5-isopropylamino--THF moiety of GRL-057-14 forms additional polar interactions with the active site of HIV-1 protease. Moreover, GRL-057-14's P1--fluoro-methylbenzene forms strong hydrogen bonding and effective van der Waals interactions. The present data suggest that the combination of C-5-aminoalkyl--THF, P1--fluoro-methylbenzene, and P2'-Cp-Abt confers highly potent activity against wild-type and multi-PI-resistant HIV strains and warrant further development of the three PIs, in particular, that of GRL-057-14, as potential therapeutic for HIV-1 infection and AIDS.
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http://dx.doi.org/10.1128/AAC.00372-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658756PMC
August 2019

Novel Central Nervous System (CNS)-Targeting Protease Inhibitors for Drug-Resistant HIV Infection and HIV-Associated CNS Complications.

Antimicrob Agents Chemother 2019 07 24;63(7). Epub 2019 Jun 24.

Department of Infectious Diseases, Kumamoto University School of Medicine, Kumamoto, Japan

There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5--fluorophenyl or P1--monofluorophenyl ring, and P2--tetrahydrofuran (-THF) or P2-tetrahydropyrano-tetrahydrofuran (-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC], 0.0002 to ∼0.003 μM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC, 0.002 to ∼0.047 μM) that had been selected to propagate at high concentrations of conventional HIV-1 PIs. Such CNS-targeting PIs maintained their antiviral activity against HIV-2 as well as multidrug-resistant clinical HIV-1 variants isolated from AIDS patients who no longer responded to existing antiviral regimens after long-term therapy. Long-term drug selection experiments revealed that the emergence of resistant-HIV-1 against these CNS-targeting PIs was substantially delayed. In addition, the CNS-targeting PIs showed the most favorable CNS penetration properties among the tested compounds, including various FDA-approved anti-HIV-1 drugs, as assessed with the blood-brain barrier reconstruction system. Crystallographic analysis demonstrated that the bicyclic rings at the P2 moiety of the CNS-targeting PIs form strong hydrogen-bond interactions with HIV-1 protease (PR) active site. Moreover, both the P1-3,5--fluorophenyl and P1--monofluorophenyl rings sustain greater van der Waals contacts with PR than in the case of darunavir (DRV). The data suggest that the present CNS-targeting PIs have desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 strains and might serve as promising preventive and/or therapeutic candidates for HIV-1-associated neurocognitive disorders (HAND) and other CNS complications.
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http://dx.doi.org/10.1128/AAC.00466-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591644PMC
July 2019

Enantioselective Total Synthesis of (+)-Monocerin, a Dihydroisocoumarin Derivative with Potent Antimalarial Properties.

J Org Chem 2019 05 29;84(10):6191-6198. Epub 2019 Apr 29.

Department of Chemistry and Department of Medicinal Chemistry , Purdue University , 560 Oval Drive , West Lafayette , Indiana 47907 , United States.

We describe here the enantioselective synthesis of (+)-monocerin and its acetate derivative. The present synthesis features an efficient optically active synthesis of the β-hydroxy-γ-lactone derivative with high enantiomeric purity using Sharpless dihydroxylation as the key step. The synthesis also highlights a tandem Lewis acid-catalyzed, oxocarbenium ion-mediated diastereoselective syn-allylation reaction, and a methoxymethyl group promoted methylenation reaction. We investigated this reaction with a variety of Lewis acids. A selective CrO-mediated oxidation of isochroman provided the corresponding lactone derivative. The synthesis is quite efficient and may be useful for the preparation of derivatives.
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http://dx.doi.org/10.1021/acs.joc.9b00414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637414PMC
May 2019

Halogen Bond Interactions of Novel HIV-1 Protease Inhibitors (PI) (GRL-001-15 and GRL-003-15) with the Flap of Protease Are Critical for Their Potent Activity against Wild-Type HIV-1 and Multi-PI-Resistant Variants.

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

Department of Refractory Viral Infections, National Center for Global Health and Medicine Research Institute, Tokyo, Japan

We generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2'-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2' ligands, respectively. GRL-001-15 and GRL-003-15 have -monofluorophenyl and -monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (ECs) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CCs) of 38 and 11 μM, respectively. The activity of GRL-001-15 against multi-PI-resistant HIV-1 variants was generally greater than that of GRL-003-15. The EC of GRL-001-15 against an HIV-1 variant that was highly resistant to multiple PIs, including darunavir (DRV) (HIV-1), was 0.17 nM, and that of GRL-003-15 was 3.3 nM, while DRV was much less active, with an EC of 216 nM. The emergence of HIV-1 variants resistant to GRL-001-15 and GRL-003-15 was significantly delayed compared to that of variants resistant to selected PIs, including DRV. Structural analyses of wild-type protease (PR) complexed with the novel PIs revealed that GRL-001-15's -fluorine atom forms halogen bond interactions (2.9 and 3.0 Å) with Gly49 and Ile50, respectively, of the protease flap region and with Pro81' (2.7 and 3.2 Å), which is located close to the protease active site, and that two fluorine atoms of GRL-142-13 form multiple halogen bond interactions with Gly49, Ile50, Pro81', Ile82', and Arg8'. In contrast, GRL-003-15 forms halogen bond interactions with Pro81' alone, suggesting that the reduced antiviral activity of GRL-003-15 is due to the loss of the interactions with the flap region.
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http://dx.doi.org/10.1128/AAC.02635-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535520PMC
June 2019