A complete structure-activity relationship of Mac1 of SARS-CoV-2, that was used to design potent nucleotide inhibitors, were reported, which were developed into membrane-permeable, non-toxic prodrugs, which strongly suppress viral replication.
Abstract
Enzymatically active macrodomains of (+)ss-RNA viruses mediate immune evasion by countering ADP-ribosylation and are therefore promising druggable targets. Here we report testing of ADP / ADP-ribose analogues for their ability to inhibit Mac1 of SARS-CoV-2, measurement of the affinity of active compounds and characterization of their binding mode by cocrystallization, uncovering critical molecular determinants of protein-ligand interaction. Key findings of the resulting structure-activity relationship (SAR) include that inhibitory potency is improved by either replacing the distal ribose of ADP-ribose by a small alkyl group or the adenine N7 by carbon. Based on insights from the SAR, we show β-methyl-GS-441524-diphosphate as nanomolar inhibitor that exhibits >1000-fold selectivity over human MacroD1 and MacroD2. Addition of C11-acyloxybenzyl (AB)-masking groups yields a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC50 0.06 µM) while exhibiting low cytotoxicity (CC50 > 50 µM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increases stability of the prodrug with little effect on toxicity and antiviral potency (EC50 = 0.03 µM), making it a membrane-permeable nucleotide-based prodrug against viral macrodomains. The authors report a complete structure-activity relationship of Mac1 of SARS-CoV-2, that was used to design potent nucleotide inhibitors. Hits were developed into membrane-permeable, non-toxic prodrugs, which strongly suppress viral replication.
Viral proteases represent validated targets for direct-acting antivirals and the treatment of associated infections. In co-crystal structures of Mpro of SARS-CoV-2 with peptidomimetic inhibitors, we noticed a spatial proximity of sidechains filling the S1’ and S2 pockets, as well as those filling S3 and S1 pockets. To enhance molecular rigidity, the proximal residues were conformationally fixed by macrocyclization. We report the synthesis of two macrocyclic series, i.e. exocyclic nitriles with linked P3 and P1 residues and endocyclic α-ketoamides with linked P1’ and P2 residues, and characterize their binding modes and bioactivities. The 17-membered macrocyclic α-ketoamide 20 f inhibited Mpro (IC₅₀ = 370 nM) and exerted anti-SARS-CoV-2 effects (EC₅₀ = 1.9 μM). Leveraging structural similarities between Mpro and the 3Cpro of enterovirus D68, we describe with two co-crystal structures how α-ketoamide macrocycles bound to and inhibited the enteroviral protease. Notably, 20 f exhibited very potent antiviral activities with EC₅₀‘s of 33, 133, and 146 nM against EV-D68, EV-A71, and CVB3, respectively. The study demonstrates how broad-spectrum activity can be achieved with direct-acting antivirals. Viral proteases are key targets for developing direct-acting antivirals to combat infections like SARS-CoV-2. Here, the authors synthesize macrocyclic α-ketoamide inhibitors, demonstrating potent inhibition of SARS-CoV-2 and enteroviruses, highlighting macrocyclization’s potential to enhance antiviral efficacy and achieve broad-spectrum activity.
R. Akula, Haifa El Kilani, Alina Metzen et al.· Communications Chemistry· 0 citations
Abstract The ongoing prevalence of SARS-CoV-2 variations highlights the urgent need for novel antiviral agents targeting key viral proteins. The papain-like protease (PLpro) is pivotal in viral replication and immune evasion, rendering it a compelling therapeutic target. In the present study, a series of novel thiadiazole- and oxadiazole-based derivatives (12-g & 13a-g) were rationally designed based on the structural features of the known PLpro inhibitor GRL0617. Convergent synthetic strategy was employed to synthesize the target compounds involving the construction of 1,3,4-thiadiazole/oxadiazole intermediates followed by amide coupling with a naphthyl-containing scaffold. All compounds were characterized by 1H NHMR, 13C NMR and mass spectrometry and evaluated for their in vitro SARS-CoV-2 PLpro inhibitory activity. Several compounds exhibited potent inhibition, surpassing the reference inhibitor GRL0617 (IC50 = 2.4 ± 1.1 µM). Among them, oxadiazole derivative (13c) featuring 3-cyanophenyl substitution emerged as the most potent inhibitor with IC50 value of 0.06 ± 0.8 µM, followed by compounds (12b, IC50 = 0.3 ± 0.3 µM), (12e, IC50 = 0.4 ± 1.6 µM) and (13e, IC50 = 0.6 ± 0.8 µM) respectively. Structure activity relationship (SAR) analysis revealed that electron-withdrawing substituents, particularly cyano group at meta position, significantly enhanced PLpro inhibition whereas methoxy/methyl groups resulted in reduced inhibition. Furthermore, molecular docking studies demonstrated favorable binding interactions of the compounds within the PLpro catalytic pocket through H-bonding, hydrophobic and π–π interactions. Moreover, molecular dynamic simulations confirmed the stability of the ligand-protein complexes throughout the simulation period supporting the experimental findings. The integrated rational design-based synthesis, biological evaluation, and computational investigations collectively identified thiadiazole/oxadiazole scaffolds as promising candidates for the development of SARS-CoV-2 inhibitors, offering valuable insights for next-generation antiviral agents targeting coronavirus proteases.
Rafaqat Hussain, Hina Sarfraz, T. Chohan et al.· Pure and Applied Chemistry· 0 citations
Mac1 is a conserved macrodomain enzyme in the nonstructural protein 3 (Nsp3) of SARS-CoV-2 and is part of the viral replication machinery. Mac1 is a target for small-molecule inhibitors that could ultimately enable new COVID-19 therapeutics to be developed. Here, we report the structure-guided design, synthesis, and Mac1 inhibition profiling of 25 analogues derived from a hit identified through crystallographic fragment screening. The heteroaryl group and scaffold (cis- and trans-cyclopentane and cyclopentene) were varied. Two new approaches to trans-cyclopentanes were developed: MacMillan’s Ir/Ni-mediated photoredox cross-coupling of alcohols and Barluenga–Valdés’ metal-free cross-coupling of sulfonyl hydrazones and boronic acids. X-ray crystal structures of 19 compounds bound to Mac1 were determined to guide the design and to rationalize the observed SAR. A new family of Mac1 inhibitors with benzothiazole or amino benzothiazoles was discovered and characterized, with IC50 values of 6–8 μM and ligand efficiency values of up to 0.40.
Xinyu Wang, William T W Butler, James R Donald et al.· Journal of Medicinal Chemist...· 0 citations
The COVID-19 pandemic severely threatened global public health, and the SARS-CoV-2 main protease (3CLpro) is a vital target for anti-COVID-19 drug development. Nevertheless, most reported 3CLpro orthosteric inhibitors readily trigger viral drug resistance, limiting their long-term clinical application. Herein, we identified a novel 3CLpro allosteric site AS1 via AlloSite and AlloReverse tools. Through virtual screening and structural optimization, a series of potent 3CLpro allosteric derivatives were designed and synthesized. FRET assays confirmed that derivatives E42 (IC50 = 59.85 ± 4.10 μM) and E60 (IC50 = 7.64 ± 0.25 μM) possessed superior inhibitory potency to the lead compound E0. Structure-activity relationship and molecular docking analyses revealed that the p-trifluoromethylphenethylamine fragment, trimethylene linker, amide bond, methyl group and 4-nitropyrazole ring are key active structural motifs, enabling E42 to specifically bind the AS1 allosteric site. The antiviral activity and cytotoxicity of E42 and E60 were evaluated using a SARS-CoV-2 trans-complementation cell culture system (containing mNG/Fluc) and the CCK-8 assay, respectively. The luciferase reporter assay revealed a consistent trend in antiviral activity among nirmatrelvir, E42, and E60, with E42 exhibiting moderate potency (EC50 = 13.79 ± 1.28 μM). Molecular dynamics simulations indicated that E42 inhibits 3CLpro activity by stabilizing its inactive conformation and disrupting protein allosteric equilibrium. Compared with conventional orthosteric inhibitors prone to drug resistance and poorly active, unsafe reported allosteric inhibitors, the optimized compounds in this study combine favorable potency and biosafety. This work provides novel lead structures and mechanistic insights for developing anti-COVID-19 3CLpro allosteric inhibitors.
SARS-CoV-2 main protease (MPro) is a proven target for drug discovery of small-molecule antiviral agents due to its crucial role in viral polyprotein processing, high structural conservation across numerous divergent variants, and the lack of similar human enzymes. Unlike covalent compounds, noncovalent inhibitors of MPro do not modify the enzyme's active site, and may offer improved safety profiles, greater chemical tractability, and better oral bioavailability without the need for pharmacokinetic enhancement. In this study, we designed, synthesized and characterized thirteen noncovalent nonpeptidic SARS-CoV-2 MPro inhibitors clustered into two series (KK and KB) of compounds. The inhibitors were designed based on our recently discovered Mcule-5948770040 and its analogue HL-3-68 designed through the structure–activity relationship study. To obtain atomic details of the inhibitors' binding we solved room-temperature X-ray structures of the MPro/inhibitor complexes, and to quantify their binding and antiviral properties we performed in vitro DSF and ITC measurements and TCID50 antiviral assays. In addition, a room-temperature neutron structure of the MPro/KB-5 complex allowed direct determination of hydrogen positions, mapping intermolecular interactions and directly visualizing the protonation states and hydrogen bonding. Improved binding affinities of KK-7 and KB-3 through KB-6 could be attributed to the observed nonconventional S–H⋯F hydrogen bond and an additional conventional hydrogen bond between the carboxamide moieties and Q189. Our study provides binding details for the designed compounds and demonstrates the feasibility of our joint X-ray/neutron structure-assisted drug design approach to generate more potent noncovalent nonpeptidic MPro inhibitors.
D. Bhandari, Katerina Kovalevskaya, Leighton Coates et al.· RSC Medicinal Chemistry· 0 citations
Structurally optimized triazine-based nucleoside analogues are identified as potential SARS-CoV-2 Mpro inhibitors and the utility of an integrated computational strategy for the rational design and prioritization of antiviral candidates for further experimental validation is demonstrated.
Madhu Sudan, Sarika Arora, Mohd Saeed et al.· Journal of the Iranian Chemi...· 0 citations