Non-covalent Inhibitors Research Articles

Machine-Learning Approach to Identify Potential Dengue Virus Protease Inhibitors: A Computational Perspective.

The global prevalence of dengue virus (DENV), a widespread flavivirus, has led to varied epidemiological impacts, economic burdens, and health consequences. The alarming increase in infections is exacerbated by the absence of approved antiviral agents against the DENV. Within flaviviruses, the NS3/NS2B serine protease plays a pivotal role in processing the viral polyprotein into distinct components, making it an attractive target for antiviral drug development. In this study, machine-learning (ML) techniques were employed to build predictive models for the screening of a library containing 32,000 protease inhibitors. Utilizing GNINA for structure-based virtual screening, the top potential candidates underwent a subsequent evaluation of their absorption, distribution, metabolism, excretion, and toxicity properties. Selected compounds were subjected to molecular dynamics simulations and binding free energy calculations via MM/GBSA. The results suggest that comp530 possesses binding potential to DENV protease as a noncovalent inhibitor with multiple positions for chemical substitutions, presenting opportunities for optimizing their selectivity and specificity. However, other compounds predicted via ML models may still provide a promising start for covalent inhibitors.

Rational Design of Macrocyclic Noncovalent Inhibitors of SARS-CoV-2 Mpro from a DNA-Encoded Chemical Library Screening Hit That Demonstrate Potent Inhibition against Pan-Coronavirus Homologues and Nirmatrelvir-Resistant Variants.

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (Mpro) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent Mpro inhibitor was the first such approved therapy. Although Mpro inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent Mpro inhibitor 5, which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone 12 with significantly improved antiviral activity. Further optimization led to the potent lactam 26, which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.

Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of N-Propargylglycine.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ1-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

Structure-Guided Conformational Restriction Leading to High-Affinity, Selective, and Cell-Active Tetrahydroisoquinoline-Based Noncovalent Keap1-Nrf2 Inhibitors.

Inhibition of the protein-protein interaction between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) has been recognized as an attractive approach for treating oxidative stress-related diseases. Here, we present a new series of noncovalent Keap1-Nrf2 inhibitors developed by a conformational restriction strategy of our fluorenone-based compounds previously identified by fragment-based drug discovery. The design was guided by X-ray cocrystal structures, and the subsequent optimization process aimed at improving affinity, cellular activity, and metabolic stability. From the noncyclic compound 7 (Ki = 2.9 μM), a new series of tetrahydroisoquinoline-based Keap1 inhibitors with up to 223-fold improvement in binding affinity (57, Ki = 13 nM), better metabolic stability, and enhanced cellular activity was obtained. In addition, the compounds showed selectivity for the Keap1 Kelch domain across a panel of 15 homologous proteins. We thereby demonstrate the utility of cyclic rigidification in the design of potent and more drug-like Keap1-Nrf2 inhibitors.

KRAS, a New Target for Precision Medicine in Colorectal Cancer?

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, with significant public health concerns. This review examines the landscape of KRAS inhibition in colorectal cancer (CRC), focusing on recent advances in therapeutic strategies targeting this oncogene. Historically deemed undruggable due to its complex structure and essential role in tumorigenesis, KRAS mutations are prevalent in CRC and are associated with poor prognosis. However, breakthroughs in drug development have led to the emergence of KRAS inhibitors as promising treatment options. This review discusses various classes of KRAS inhibitors, including covalent and non-covalent inhibitors, as well as combination therapies aimed at enhancing efficacy and overcoming resistance mechanisms. It highlights recent clinical trials evaluating the efficacy of KRAS inhibitors either as monotherapy or in combination with other agents, such as anti-EGFR antibodies. Despite challenges such as resistance mechanisms and tumor heterogeneity, the development of KRAS inhibitors represents a significant advance in CRC treatment and holds promise for improving patient outcomes in the future.

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors.

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (Kdimer) 2-3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ∼10 to 30%, relative to that of the wild-type enzyme, without altering Kdimer. Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MProWT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MProWT-ensitrelvir. Chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

Thiol-Reactive or Redox-Active: Revising a Repurposing Screen Led to a New Invalidation Pipeline and Identified a True Noncovalent Inhibitor Against Papain-like Protease from SARS-CoV-2

Thiol-Reactive or Redox-Active: Revising a Repurposing Screen Led to a New Invalidation Pipeline and Identified a True Noncovalent Inhibitor Against Papain-like Protease from SARS-CoV-2

Novel Alkynylamide-Based Nonpeptidic Allosteric Inhibitors for SARS-CoV-2 3-Chymotrypsin-like Protease.

Although the coronavirus disease 2019 (COVID-19) crisis has passed, there remains a necessity for continuous efforts toward developing more targeted drugs and preparing for potential future virus attacks. Currently, most of the drugs received authorization for the treatment of COVID-19 have exhibited several limitations, such as poor metabolic stability, formidable preparation, and uncertain effectiveness. It is still significant to develop novel, structurally diverse small-molecule antiviral drugs targeting SARS-CoV-2 3-chymotrypsin-like protease (3CLpro). Herein, we report a class of alkynylamide-based nonpeptidic 3CLpro inhibitors that can be prepared conveniently by our previously developed one-pot synthetic method. The structure-activity relationships of alkynylamides as SARS-CoV-2 3CLpro inhibitors have been carefully investigated and discussed in this study. The two stereoisomers of the resulting molecules exhibit stereoselective interaction with 3CLpro, and the optimized compound (S,R)-4y inhibits 3CLpro with high potency (IC50 = 0.43 μM), low cytotoxicity, and acceptable cell permeability. Compound (S,R)-4y presents as a noncovalent inhibitor of 3CLpro against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. The Lineweaver-Burk plots, binding energy, surface plasmon resonance, and molecular docking studies suggest that (S,R)-4y specifically binds to an allosteric pocket of the SARS-CoV-2 3CLpro. These findings provide a novel class of nonpeptidic alkynylamide-based allosteric inhibitors with high selectivity against SARS-CoV-2 3CLpro featured by a simplified one-pot synthesis at room temperature in air.

Exploring novel antitubercular agents: Innovative design of 2,3-diaryl-quinoxalines targeting DprE1 for effective tuberculosis treatment

Abstract The rising instances of drug resistance in Mycobacterium tuberculosis strains pose a significant global health challenge. Conventional tuberculosis (TB) treatments, which typically involve multiple antibiotics, face hurdles like drug resistance, reduced effectiveness, and heightened toxicity. Consequently, there is a pressing need for innovative anti-TB agents with new modes of action. Decaprenyl-phosphoryl-β-d-ribose 2′-epimerase 1(DprE1), a crucial enzyme in Mycobacterium tuberculosis, plays a vital role in cell wall biosynthesis – a critical aspect for the bacterium’s survival. Building on the success of diarylquinolines like bedaquiline, targeting DprE1 presents a promising avenue for developing anti-TB drugs, especially against drug-resistant strains. Our research focused on discovering novel DprE1 inhibitors using a ligand-based drug design strategy, starting with the established non-covalent inhibitor Ty38c. We assembled a library of 16 molecules, modifying them based on factors like drug-like properties, chemical accessibility, and synthetic feasibility. Molecular docking analyses of this library identified three molecules with binding affinities comparable to Ty38c. Among these, KS_QD_05 and KS_QD_04 are promising candidates, which were further validated through molecular dynamics simulation studies where root-mean-square deviation (RMSD) values of all three complexes reached a plateau, measuring around 0.3 nm, indicating that the apoprotein and all complexes stabilized during the simulation. The ligands KS_QD_04 and KS_QD_05 displayed significantly stable deviation. KS_QD_05 reached about 0.1 nm equilibrium value. However, the ligand KS_QD_04 reached an RMSD value of 0.17 nm and showed distress at 70 nm. KS_QD_04 and KS_QD_05 showed an average value of 1-3 H-bond interaction and regarding the RMSF values, both the compounds showed fluctuations less than 0.5 nm in the case of Mtb. DprE1 enzyme. This indicates the potential of both compounds to become lead compounds in the pursuit of DprE1 inhibitors for TB treatment.

151 (PB139): CFON-026, a best-in-class macrocyclic non-covalent inhibitor of BTK (WT) and all clinically relevant BTK resistance mutations

151 (PB139): CFON-026, a best-in-class macrocyclic non-covalent inhibitor of BTK (WT) and all clinically relevant BTK resistance mutations

C-4 analogues of podophyllotoxin as tubulin inhibitors: synthesis, biological evaluation, and structure-activity relationship

The diversity of lignan small molecules derived from podophyllotoxin, a non-covalent tubulin inhibitor isolated from the Podophyllum family, has led to the clinical development of FDA-approved anticancer agents etoposide and teniposide. While these two compounds share the same tetracyclic core as podophyllotoxin, two subtle structural changes—4’ demethylation on the aromatic ring and stereospecific glycosylation at the C-4 hydroxyl—result in an alternate biological mechanism. Given the immense pharmacological importance of altering the C-4 position, we synthesised and evaluated a systematic library of diversified esters to establish a structure-activity relationship regarding modification at C-4 on the properties of podophyllotoxin. We determined the biological activity of these esters through cell viability assays, computer docking models, tubulin polymerisation assays, and cell cycle analysis. Altogether, we demonstrate that increasing steric hindrance at C-4 leads to a loss in potency against human cancer cells but has a significantly lesser impact on cell-free tubulin inhibition.

Machine Learning and Deep Learning Models for Predicting Noncovalent Inhibitors of AmpC β-Lactamase.

Continuous use of antibiotics leads to the ability of bacteria to adapt by developing complex antibiotic resistance (AR) mechanisms. The synthesis of β-lactamases is a widely observed AR mechanism. The class C β-lactamase (AmpC) causes significant resistance toward β-lactam antibiotics, and new treatments are urgently needed. Noncovalent inhibitors have been developed against a broad spectrum of β-lactamases. In this study, we developed robust and accurate models for predicting noncovalent inhibitors of AmpC using large compound data sets and machine/deep learning modeling. We created support vector machine (SVM), random forest (RF), and feed-forward neural network (FFNN) classification models. The cross-validation (CV) accuracies varied between 80 and 82%, as combined models reached an accuracy of 83%. We analyzed the physicochemical characteristics of the noncovalent inhibitors and predicted the binding modes for some of them. Such models are helpful for identifying new noncovalent inhibitors in order to establish novel solutions against the growing resistance to standard β-lactam inhibitors. The best RF, SVM, and FFNN models for predicting noncovalent inhibitors of AmpC β-lactamase are available in the GitHub repository, https://github.com/UPCmctr/ML-DL-AmpC-B-lactamase.

Advances in the Search for SARS-CoV-2 Mpro and PLpro Inhibitors.

SARS-CoV-2 is a spherical, positive-sense, single-stranded RNA virus with a large genome, responsible for encoding both structural proteins, vital for the viral particle's architecture, and non-structural proteins, critical for the virus's replication cycle. Among the non-structural proteins, two cysteine proteases emerge as promising molecular targets for the design of new antiviral compounds. The main protease (Mpro) is a homodimeric enzyme that plays a pivotal role in the formation of the viral replication-transcription complex, associated with the papain-like protease (PLpro), a cysteine protease that modulates host immune signaling by reversing post-translational modifications of ubiquitin and interferon-stimulated gene 15 (ISG15) in host cells. Due to the importance of these molecular targets for the design and development of novel anti-SARS-CoV-2 drugs, the purpose of this review is to address aspects related to the structure, mechanism of action and strategies for the design of inhibitors capable of targeting the Mpro and PLpro. Examples of covalent and non-covalent inhibitors that are currently being evaluated in preclinical and clinical studies or already approved for therapy will be also discussed to show the advances in medicinal chemistry in the search for new molecules to treat COVID-19.

Discovery, Characterization, and Structure of a Cell Active PAD2 Inhibitor Acting through a Novel Allosteric Mechanism.

Peptidyl arginine deiminases (PADs) are important enzymes in many diseases, especially those involving inflammation and autoimmunity. Despite many years of effort, developing isoform-specific inhibitors has been a challenge. We describe herein the discovery of a potent, noncovalent PAD2 inhibitor, with selectivity over PAD3 and PAD4, from a DNA-encoded library. The biochemical and biophysical characterization of this inhibitor and two noninhibitory binders indicated a novel, Ca2+ competitive mechanism of inhibition. This was confirmed via X-ray crystallographic analysis. Finally, we demonstrate that this inhibitor selectively inhibits PAD2 in a cellular context.

Enzyme Dynamics Determine the Potency and Selectivity of Inhibitors Targeting Disease-Transmitting Mosquitoes.

Vector control of mosquitoes with insecticides is an important tool for preventing the spread of mosquito-borne diseases including malaria, dengue, chikungunya, and Zika. Development of active ingredients for insecticides are urgently needed because existing agents exhibit off-target toxicity and are subject to increasing resistance. We therefore seek to develop noncovalent inhibitors of the validated insecticidal target acetylcholinesterase 1 (AChE1) from mosquitoes. Here we use molecular dynamics simulations to identify structural properties essential for the potency of reversible inhibitors targeting AChE1 from Anopheles gambiae (AgAChE1), the malaria-transmitting mosquito, and for selectivity relative to the vertebrate Mus musculus AChE (mAChE). We show that the collective motions of apo AgAChE1 and mAChE differ, with AgAChE1 exhibiting less dynamic movement. Opening and closing of the gorge, which regulates access to the catalytic triad, is enabled by different mechanisms in the two species, which could be linked to their differing amino acid sequences. Inhibitor binding reduced the overall magnitude of dynamics of AChE. In particular, more potent inhibitors reduced the flexibility of the Ω loop at the entrance of the gorge. The selectivity of inhibitors for AgAChE1 over mAChE derives from the positioning of the α-helix lining the binding gorge. Our findings emphasize the need to consider dynamics when developing inhibitors targeting this enzyme and highlight factors needed to create potent and selective AgAChE1 inhibitors that could serve as active ingredients to combat disease-transmitting mosquitoes.

Abstract PR-16: Evaluating direct KRASQ61H inhibition in pancreatic cancer models

Abstract KRASQ61 mutations are associated with the worst prognosis among the various KRAS mutational subtypes of pancreatic ductal adenocarcinoma (PDAC). RMC-7977 is a RAS(ON) multi-selective noncovalent inhibitor with broad spectrum activity against oncogenic RAS mutants and wild-type RAS. The related investigational agent RMC-6236 is currently undergoing clinical trials. Binary complexes of CypA: RMC-7977 bind equipotently to multiple KRAS mutational subtypes however RMC-7977 is less potent at inhibiting growth in KRASQ61H-mutant cell lines. In contrast, RM-047 is a preclinical RAS(ON) Q61H-selective inhibitor tool compound. We evaluated direct KRASQ61H inhibition using both RMC-7977 and RM-047 in cell culture models. We found that in KRASQ61H-rescued RASless mouse embryonic fibroblasts (MEFs), RM-047 inhibited ERK activity and suppressed growth more potently than RMC-7977. Conversely, in a panel of KRASQ61H-mutant PDAC cell lines, RM-047 was less potent than RMC-7977 in two out of three KRASQ61H-mutant PDAC cell lines. Furthermore, we discovered that ERK activity rapidly rebounded with treatment in KRASQ61H-mutant PDAC cell lines, whereas ERK activity remained suppressed in KRASQ61H-rescued RASless MEFS lacking WT RAS alleles. Our analysis of DepMap data indicated that among the KRAS mutational subtypes frequently found in pancreatic cancer, KRASQ61H is the least dependent on KRAS for survival. This suggests that in a KRASQ61H-mutant PDAC setting, additional factors such as WT RAS expression may mediate growth. From our KRAS drug-anchored CRISPR loss-of-function screens, we found that the loss of EGFR sensitized cells to KRAS inhibition, particularly in a KRASQ61H-dependent context. Therefore, we combined erlotinib, an FDA-approved EGFR inhibitor, with either RM-047 or RMC-7977, and found that the addition of erlotinib prevented ERK rebound activity and synergized with RM-047 or RMC-7977 to reduce growth in KRASQ61H-mutant PDAC cell lines. These data provide insights into mechanisms by which KRASQ61H-mutant PDAC may be less dependent on KRAS for survival and further support the clinical evaluation of combined EGFR and RAS(ON) inhibition as a potential therapeutic strategy in the treatment of PDAC patients harboring KRASQ61H mutations. Citation Format: Szu-Aun Long, Amber M Amparo, Haley Todd, Grace Goodhart, Syed A Ahmad, Andrew M Waters. Evaluating direct KRAS inhibition in pancreatic cancer models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-16.

Discovery of new non-covalent reversible BTK inhibitors: Synthesis, in silico studies, and in vitro evaluations

Bruton's Tyrosine Kinase (BTK) played a key role in the B cell antigen receptor (BCR) signaling pathway, and was considered a hotspot in the treatment of B cell malignant tumors and B cell immune diseases. There were 5 covalent irreversible inhibitors launched currently on the market, but C481S mutation was detected in most patients after administration. The approval of Pirtobrutinib (Jaypirca) by FDA in 2023 aroused great interest in the development of non-covalent and reversible BTK inhibitors. In order to solve the resistance of covalent irreversible inhibitors caused by C481S mutation, 11 reversible BTK inhibitors were designed based on screening in this article. The design, synthesis, in silico studies, and in vitro evaluations were performed for further verification. Among them, compound WS-11 showed best activity with IC50 of 3.9 nM for wild type, 2.2 nM for C481S mutation BTK, which was comparable to the positive control Pirtobrutinib. Furthermore, WS-11 would have a good druglikeness properties predicted by pkCSM and SwissADME, which provided a promising lead for further optimization and development.

Development and Evaluation of Bis-benzothiazoles as a New Class of Benzothiazoles Targeting DprE1 as Antitubercular Agents.

Benzothiazole-bearing compounds have emerged as potential noncovalent DprE1 (decaprenylphosphoryl-β-d-ribose-2'-epimerase) inhibitors active against Mycobacterium tuberculosis. Based on structure-based virtual screening (PDB ID: 4KW5), a focused library of thirty-one skeletally diverse benzothiazole amides was prepared, and the compounds were assessed for their antitubercular activity against M.tb H37Ra. Most potent compounds 3b and 3n were further evaluated against the M.tb H37Rv strain by the microdilution assay method. Among the compounds evaluated, bis-benzothiazole amide 3n emerged as a hit molecule and demonstrated promising antitubercular activity with minimum inhibitory concentration (MIC) values of 0.45 μg/mL and 8.0 μg/mL against H37Ra and H37Rv, respectively. Based on the preliminary hit molecule (3n), a focused library of 12 more bis-benzothiazole amide derivatives was further prepared by varying the substituents on either side to obtain new leads and generate a structure-activity relationship (SAR). Among these compounds, 6a, 6c, and 6d demonstrated remarkable antitubercular activity with MIC values of 0.5 μg/mL against H37Ra and 1.0, 2.0, and 8.0 μg/mL against H37Rv, respectively. The most active compound, 6a, also displayed significant efficacy against four drug-resistant tuberculosis strains. Compound 6a was assessed for in vitro cytotoxicity against the HepG2 cell line, and it displayed insignificant cytotoxicity. Furthermore, time-kill kinetic studies demonstrated time- and dose-dependent bactericidal activity of this compound. The GFP release assay revealed that compound 6a targets the inhibition of a cell wall component. SNPs in dprE-1 gene assessment revealed that compound 6a binds to tyrosine at position 314 of DprE1 and replaces it with histidine, causing resistance similar to that of standard TCA1. In silico docking studies further suggest that the strong noncovalent interactions of these compounds may lead to the development of potent noncovalent DprE1 inhibitors.

Correlating Predicted Reactivities with Experimental Inhibition Data of Covalent ChlaDUB1 Inhibitors.

Covalent inhibition with electrophiles of suitable reactivity allows for prolonged inactivation of a targeted enzyme, in comparison to noncovalent inhibitors. To fine-tune covalently reacting groups, various computational workflows have been developed for reactivity prediction. Their results are typically evaluated based on in vitro assays that use cysteine, glutathione, or other model nucleophiles, but the relevance of the predictions for the situation in a particular enzyme environment remains under-investigated. Herein, we utilized a DFT-based computational method to assess the transferability of predicted reactivities to enzyme inhibition. For this, we designed and synthesized a set of covalent inhibitors of ChlaDUB1, a potential target to fight the pathogen Chlamydia trachomatis. We show that in the context of enzyme inhibition, geometrical congruence between ligands has to be taken into account when evaluating the reactivity of covalently reacting groups in respective drug discovery projects.

Structure and Inhibition of Insect UDP-N-acetylglucosamine Pyrophosphorylase: A Key Enzyme in the Hexosamine Biosynthesis Pathway.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.