N-myristoyltransferase Research Articles

Discovery of Novel Myristic Acid Derivatives as N-Myristoyltransferase Inhibitors: Design, Synthesis, Analysis, Computational Studies and Antifungal Activity.

In recent years, N-Myristoyltransferase (NMT) has been identified as a new target for the treatment of fungal infections. It is observed that at present, there are increased rates of morbidity and mortality due to fungal infections. Hence, a series of novel myristic acid derivatives were designed via molecular docking studies and ADMET studies by targeting NMT (N-Myristoyltransferase). The designed myristic acid derivatives were synthesized by converting myristic acid into myristoyl chloride and coupling it with aryl amines to yield corresponding myristic acid derivatives. The compounds were purified and characterized via FTIR, NMR and HRMS spectral analyses. In this study, we carried out a target NMT inhibition assay. In the NMT screening assay results, the compounds 3u, 3m and 3t showed better inhibition compared to the other myristic acid derivatives. In an in vitro antifungal evaluation, the myristic acid derivatives were assessed against Candida albicans and Aspergillus niger strains by determining their minimal inhibitory concentrations (MIC50). The compounds 3u, 3k, 3r and 3t displayed superior antifungal capabilities against Candida albicans, and the compounds 3u, 3m and 3r displayed superior antifungal capabilities against Aspergillus niger compared to the standard drug FLZ (fluconazole). Altogether, we identified a new series of antifungal agents.

A first-in-human, open-label, phase I trial of daily oral PCLX-001, an NMT inhibitor, in patients with relapsed/refractory B-cell lymphomas and advanced solid tumors.

e15094 Background: Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. PCLX-001 is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC50 of 5nM and 8nM, respectively). In vitro, hematologic cancer cell lines were exquisitely sensitive to PCLX-001. PCLX-001 regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient derived xenograft model. PCLX-001 also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we hypothesized that PCLX-001 would be safe, tolerable, and active as a novel oral cancer therapeutic. Methods: Patients with relapsed/refractory B-cell lymphomas and advanced solid tumors are being enrolled in a multicenter, open label, standard phase I dose escalation design to determine feasibility, maximum tolerated dose (MTD), and pharmacokinetics of PCLX-001 monotherapy. Seven dose levels are being studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 200 mg, and 280 mg. Dose limiting toxicity (DLT) is defined as: grade (Gr) 4 platelets, Gr 3 platelets with bleeding, Gr 4 ANC ≥ 7 days, febrile neutropenia, or clinically significant > Gr 3 non-hematologic toxicity. Results: Eighteen evaluable patients (3 relapsed B-cell lymphoma; 15 advanced solid tumor) have been accrued through dose level six (200 mg cohort) without DLT. No attributable Gr 3 or 4 toxicities have been identified. Noncompartmental pharmacokinetic (PK) analyses demonstrate rapid oral absorption, a terminal half-life of approximately 10 hours, rapid achievement of steady state, and no induction of metabolism. PCLX-001 trough plasma concentrations at higher dose cohorts exceed the EC90 required to inhibit cultured cancer cells. Conclusions: PCLX-001 is safe and well tolerated up to the 200 mg daily dose. Our results support the ongoing development of PCLX-001 as an oral daily therapy for the treatment of patients with cancer. Updated study results will be presented, summarizing the safety, MTD, and PK outcomes in PCLX-001 treated patients. Preliminary clinical activity will be determined in 20 patient dose expansion cohorts of relapsed/refractory B-cell lymphomas, and selected advanced solid tumors following the identification of PCLX-001 MTD. Clinical trial information: NCT04836195 .

Pharmacophore-guided drug design using LdNMT as a model drug target for leishmaniasis

Leishmaniasis is caused by Leishmania genus parasites and has a high mortality rate. The available drugs to treat leishmaniasis fail due to acquired resistance in parasites. Several enzymes of the Leishmania parasite have been used to design new therapeutic molecules against leishmaniasis. This study uses a pharmacophore-guided approach to design the drug candidate by targeting Leishmania N-Myristoyl transferase (LdNMT). From the initial sequence analysis of LdNMT, we have identified a unique 20 amino acid stretch exploited for screening and designing the small molecules. The pharmacophore for the myristate binding site on LdNMT was elucidated, and a heatmap was constructed. The leishmanial NMT pharmacophore has similarities with other pathogenic microorganisms. Moreover, substituting alanine in pharmacophoric residues elevates the affinity of myristate with NMT. Furthermore, a molecular dynamics (MD) simulation study was conducted to ascertain the stability of the mutants and or wild type. The wild-type NMT has a comparatively low affinity to myristate compared to alanine mutants, indicating that hydrophobic residues favor the myristate binding. The molecules were initially designed by using pharmacophore as a sieving mechanism. In subsequent steps, the selected molecules screened against leishmanial unique amino acid stretch and subsequently with human, leishmanial full-size NMTs. The compounds BP5, TYI, DMU, 3PE and 4UL were the top hits and chemical features similar to the myristate. The molecule 4UL was found to be highly specific towards leishmanial NMT over human NMT, suggesting the molecule is a strong leishmanial NMT inhibitor. The molecule can be taken further to assess it in in-vitro conditions.

Identification of new oxospiro chromane quinoline-carboxylate antimalarials that arrest parasite growth at ring stage

Malaria still threatens half the globe population despite successful Artemisinin-based combination therapy. One of the reasons for our inability to eradicate malaria is the emergence of resistance to current antimalarials. Thus, there is a need to develop new antimalarials targeting Plasmodium proteins. The present study reported the design and synthesis of 4, 6 and 7-substituted quinoline-3-carboxylates 9(a–o) and carboxylic acids 10(a–b) for the inhibition of Plasmodium N-Myristoyltransferases (NMTs) using computational biology tools followed by chemical synthesis and functional analysis. The designed compounds exhibited a glide score of −9.241 to −6.960 kcal/mol for PvNMT and −7.538 kcal/mol for PfNMT model proteins. Development of the synthesized compounds was established via NMR, HRMS and single crystal X-ray diffraction study. The synthesized compounds were evaluated for their in vitro antimalarial efficacy against CQ-sensitive Pf3D7 and CQ-resistant PfINDO lines followed by cell toxicity evaluation. In silico results highlighted the compound ethyl 6-methyl-4-(naphthalen-2-yloxy)quinoline-3-carboxylate (9a) as a promising inhibitor with a glide score of −9.084 kcal/mol for PvNMT and −6.975 kcal/mol for PfNMT with IC50 values of 6.58 µM for Pf3D7 line. Furthermore, compounds 9n and 9o exhibited excellent anti-plasmodial activity (Pf3D7 IC50 = 3.96, 6.71 µM, and PfINDO IC50 = 6.38, 2.8 µM, respectively). The conformational stability of 9a with the active site of the target protein was analyzed through MD simulation and was found concordance with in vitro results. Thus, our study provides scaffolds for the development of potent antimalarials targeting both Plasmodium vivax and Plasmodium falciparum. Communicated by Ramaswamy H. Sarma

A role of salt bridges in mediating drug potency: A lesson from the N-myristoyltransferase inhibitors.

The salt bridge is the strongest non-covalent interaction in nature and is known to participate in protein folding, protein-protein interactions, and molecular recognition. However, the role of salt bridges in the context of drug design has remained not well understood. Here, we report that a common feature in the mechanism of inhibition of the N-myristoyltransferases (NMT), promising targets for the treatment of protozoan infections and cancer, is the formation of a salt bridge between a positively charged chemical group of the small molecule and the negatively charged C-terminus of the enzyme. Substituting the inhibitor positively charged amine group with a neutral methylene group prevents the formation of the salt bridge and leads to a dramatic activity loss. Molecular dynamics simulations have revealed that salt bridges stabilize the NMT-ligand complexes by functioning as molecular clips that stabilize the conformation of the protein structure. As such, the creation of salt bridges between the ligands and their protein targets may find an application as a valuable tool in rational drug design.

Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Identification of the underlying mechanism of HCC progression and exploration of new therapeutic drugs are urgently needed. Here, a compound library consisting of 419 FDA-approved drugs was taken to screen potential anticancer drugs. A series of functional assays showed that desloratadine, an antiallergic drug, can repress proliferation in HCC cell lines, cell-derived xenograft (CDX), patient-derived organoid (PDO) and patient-derived xenograft (PDX) models. N-myristoyl transferase 1 (NMT1) was identified as a target protein of desloratadine by drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) assays. Upregulation of NMT1 expression enhanced but NMT1 knockdown suppressed tumor growth in vitro and in vivo. Metabolic labeling and mass spectrometry analyses revealed that Visinin-like protein 3 (VILIP3) was a new substrate of NMT1 in protein N-myristoylation modification, and high NMT1 or VILIP3 expression was associated with advanced stages and poor survival in HCC. Mechanistically, desloratadine binds to Asn-246 in NMT1 and inhibits its enzymatic activity, disrupting the NMT1-mediated myristoylation of the VILIP3 protein and subsequent NFκB/Bcl-2 signaling. Conclusively, this study demonstrates that desloratadine may be a novel anticancer drug and that NMT1-mediated myristoylation contributes to HCC progression and is a potential biomarker and therapeutic target in HCC.

Identification of Potential Leishmania N-Myristoyltransferase Inhibitors from Withania somnifera (L.) Dunal: A Molecular Docking and Molecular Dynamics Investigation.

Leishmaniasis is a group of infectious diseases caused by Leishmania protozoa. The ineffectiveness, high toxicity, and/or parasite resistance of the currently available antileishmanial drugs has created an urgent need for safe and effective leishmaniasis treatment. Currently, the molecular-docking technique is used to predict the proper conformations of small-molecule ligands and the strength of the contact between a protein and a ligand, and the majority of research for the development of new drugs is centered on this type of prediction. Leishmania N-myristoyltransferase (NMT) has been shown to be a reliable therapeutic target for investigating new anti-leishmanial molecules through this kind of virtual screening. Natural products provide an incredible source of affordable chemical scaffolds that serve in the development of effective drugs. Withania somnifera leaves, roots, and fruits have been shown to contain withanolide and other phytomolecules that are efficient anti-protozoal agents against Malaria, Trypanosoma, and Leishmania spp. Through a review of previously reported compounds from W. somnifera-afforded 35 alkaloid, phenolic, and steroid compounds and 132 withanolides/derivatives, typical of the Withania genus. These compounds were subjected to molecular docking screening and molecular dynamics against L. major NMT. Calycopteretin-3-rutinoside and withanoside IX showed the highest affinity and binding stability to L. major NMT, implying that these compounds could be used as antileishmanial drugs and/or as a scaffold for the design of related parasite NMT inhibitors with markedly enhanced binding affinity.

Artemisia herba-alba sesquiterpenes: in silico inhibition in the ATP-binding pocket.

To identify antimicrobial leads for medical applications, metabolites from the aerial part of Artemisia herba-alba were extracted and chromatographically purified. Two new sesquiterpenes, 1β,8α-dihydroxyeudesm-4-en-6β,7α,11βH-12,6-olide (1) and 1β,6α,8α-trihydroxy, 11α-methyl-eudesma-4(15)-en-13-propanoate (2) along with a known eudesmanolide 11-epi-artapshin (3) were identified. Structures were determined by spectroscopic methods including 1D- and 2D-NMR as well as mass spectroscopy. Compound 3 inhibited Gram-positive bacteria Bacillus subtilis, Lactobacillus cereus and Staphylococcus aureus and exhibited antifungal activity against the pathogenic fungus F. solani. The mode-of-action of these antimicrobial sesquiterpenes as bacterial type II DNA topoisomerase and/or DNA gyrase B inhibitors were examined via in silico studies. Such molecular-docking studies were also employed to examine antifungal activity against an N-myristoyl transferase (NMT) target. Compound 3 had the greatest gyrase B binding affinity in the ATP-binding pocket and was found to possess an inhibitory action against non-invasive micro-test technology (NMT).

A computational approach to identify novel plant metabolites against Aspergillus fumigatus

Aspergillus fumigatus, a common saprotrophic fungus is one of the most important causative agents of invasive aspergillosis. People with compromised immune systems or lung disorders are at a higher risk of acquiring Aspergillus-related health issues. The global number of invasive aspergillosis cases is expected to be over 300000 per year, with a 50 % death rate even when recognized and treated early. Current noninvasive therapies for fungal infections are azoles. As A. fumigatus resistance to azoles is rising, this study attempted to evaluate several plant metabolites as potential inhibitory agents of A. fumigatus by using in silico method. Five essential proteins including Chitinase, Glycosyl hydrolases, Glycosylphosphatidylinositol anchor, N-myristoyl transferase, and O-mannosyltransferases were selected as target proteins due to their involvement in morphogenetic processes, cell wall polysaccharides biosynthesis, filamentation, survival, organismal characteristics, proliferation, and cell wall integrity. Out of our 20 plant metabolites, four metabolites i.e., Chlorogenic acid, Protopine, Spirostanol, and Viniferin showed the best binding affinity with the key outer membrane protein of A. fumigatus during molecular docking analysis. Viniferin showed the highest binding affinity of about −262.07 and −247.63 with Chitinase and glycosyl hydrolases respectively. Molecular dynamic simulation was used to look further into the stability of the best two protein-ligand complexes. The average RMSD value of 2.4 A° for Chitinase-Viniferin and 2.2 A° for Glycosyl hydrolases-Viniferin complex demonstrated their excellent stability. ADME (absorption, distribution, metabolism, and excretion) analysis of these four plant metabolites showed that none of them had any side effects that might interfere with their drug-likeness efficiency, and no substantial toxic effects or allergenicity were also identified. Thus, all the predicted four plant metabolites can be used as a possible inhibitor of A. fumigatus. Furthermore, we strongly recommend more in vitro and in vivo trials to experimentally strengthen our encouraging outcomes.

Inspection of in-house designed novel thiochromone amino-acid conjugate derivatives as Lm-NMT inhibitor – An in-silico analysis

Leishmaniasis is a complex neglected tropical disease caused by various leishmanial parasites that primarily affect the world's poorest people. A limited number of standard medications are available for this disease that has been used for several decades, which have drawbacks such as resistance, higher cost, and patient compliance, making it difficult to reach the poor. The search for novel chemical entities to treat leishmaniasis has led to target-based scaffold research. Thiochromone moieties in conjugation with aromatic amino acids have been considered for the study, along with possible substitutions of the electron-withdrawing and electron-donating groups. N-myristoyl transferase (NMT) has been selected as the molecular target for the study responsible for protein-protein interaction and ribosylation of proteins necessary for the growth inside the human body of the parasite. The designed novel thiochromone analogs were docked against the selected leishmanial NMT using thein-silico methods, physicochemical and toxicity properties were predicted, and Structure-Activity Relationship was also established in-silico. Finally, a molecular dynamics simulation study for 100 ns gave an idea about the stability of the protein-ligand complex. A time frame analysis of each 10 ns confirmation was also studied to understand better the putative binding pattern designed analogs.

Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins.

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

Targeting N-Myristoylation for Adult Acute Myeloid Leukemia Therapy

Protein N-myristoylation is the covalent attachment of myristic acid (C14:0) by an amide linkage to the NH2-terminal glycine residue of numerous eukaryotic and viral proteins, thereafter regulating their membrane targeting or enzymatic activity. A single myristoyl-CoA: protein N-myristoyltransferase (NMT) isoform catalyses the acylation reaction in yeast, insects, plants, and unicellular parasites. It is two separate isoforms however; NMT1 and NMT2, with predominantly overlapping substrates that perform this task in mammals. Myristoylated proteins that fail to undergo the modification are recognised by a dedicated glycine N-degron system and rapidly degraded. We recently identified a remarkable reduction of NMT2 expression in a wide variety of cancers, especially hematological cancers. As a consequence, we analysed if NMT1/NMT2 could be associated with AML progression. Data analysis from the TCGA transcriptome database showed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. One oncogenic myristoylated target identified initially in B-cell lymphoma is the family of Src kinases (SFKs). Here, we demonstrate that PCLX-001 significantly reduces total and phosphorylated SFKs, which are essential for pro-survival signalling downstream frequently mutated c-Kit and FLT3 receptors in multiple AML cell lines. Moreover, inhibiting myristoylation led to ER stress induction and apoptosis in cell lines and patient blast samples at concentrations sparing most peripheral blood lymphocytes and monocytes from healthy individuals. We further validated the therapeutic potential of our lead myristoylation inhibitor ongoing clinical trial evaluation against lymphoma and solid tumours (PCLX-001) in a variety of AML xenograft models. PCLX-001 monotherapy had a dose-dependent tumoricidal effect in an ectopic sub-cutaneous MV-4-11 xenograft model, in two tail vein injected patient derived xenografts (PDXs) as well as in 3 out of 5 intra-femoral PDXs. Of note, five days of PCLX-001 treatment at the highest concentration was sufficient to obtain complete tumour eradication in our CDX model. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow with minimal toxicity or long-term impact on body weight (Fig.1). Further analyses are required to characterize the cytogenetics, common mutations or NMTs expression profile that could explain the sensitivity of certain patient models compared to the others. An ongoing challenge in treatment of leukemia resides in leukemic stem cells (LSCs), a low-frequency sub-population functioning as progenitor cells. Without eradication of LSCs no patient can be cured. Relapse rates are high in AML and are often featuring additional mutations and drug resistance. Here, we demonstrate that LSC subpopulations (CD34+/CD38- and CD117+/ CD15-) in the OCI-AML-22 patient derived model are exquisitely sensitive to PCLX-001 treatment in vitro and began shrinking after 48 hours of treatment. In addition, the LSC subpopulation showed lower IC50 compared to the general cell population at 72 hours. Interestingly, we identified that functionally validated LSC+ patient subpopulations were expressing lower amount of NMT2 compared to LSC-. This observation could partially explain the sensitivity of the LSC subpopulation. In aggregate, our preclinical observations suggest that PCLX-001 is highly potent against AML cells in vitro and in vivo (both circulating and in the bone marrow), and, that LSCs are also more sensitive to PCLX-001 than non-leukemic stem cells. This further supports the initiation of PCLX-001evaluation in AML clinical trials scheduled to start in late 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of the Antifungal, Antioxidant, and Anti-Diabetic Potential of the Essential Oil of Curcuma longa Leaves from the North-Western Himalayas by In Vitro and In Silico Analysis.

Essential oils (EOs) have gained immense popularity due to considerable interest in the health, food, and pharmaceutical industries. The present study aimed to evaluate the antimicrobial and antioxidant activity and the anti-diabetic potential of Curcuma longa leaf (CLO) essential oil. Further, major phytocompounds of CLO were analyzed for their in-silico interactions with antifungal, antioxidant, and anti-diabetic proteins. CLO was found to have a strong antifungal activity against the tested Candida species with zone of inhibition (ZOI)-11.5 ± 0.71 mm to 13 ± 1.41 mm and minimum inhibitory concentration (MIC) was 0.63%. CLO also showed antioxidant activity, with IC50 values of 5.85 ± 1.61 µg/mL using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay and 32.92 ± 0.64 µM using ferric reducing antioxidant power (FRAP) assay. CLO also showed anti-diabetic activity with an IC50 of 43.06 ± 1.24 µg/mL as compared to metformin (half maximal inhibitory concentration, IC50-16.503 ± 0.66 µg/mL). Gas chromatography-mass spectrometry (GC-MS) analysis of CLO showed the presence of (-)-zingiberene (17.84%); 3,7-cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-(15.31%); cyclohexene, 4-methyl-3-(1-methylethylidene) (12.47%); and (+)-4-Carene (11.89%) as major phytocompounds. Molecular docking of these compounds with antifungal proteins (cytochrome P450 14 alpha-sterol demethylase, PDB ID: 1EA1, and N-myristoyl transferase, PDB ID: 1IYL), antioxidant (human peroxiredoxin 5, PDB ID: 1HD2), and anti-diabetic proteins (human pancreatic alpha-amylase, PDB ID: 1HNY) showed strong binding of 3,7-cyclodecadien-1-one with all the selected protein targets. Furthermore, molecular dynamics (MD) simulations for a 100 ns time scale revealed that most of the key contacts of target proteins were retained throughout the simulation trajectories. Binding free energy calculations using molecular mechanics generalized born surface area (MM/GBSA), and drug-likeness and toxicity analysis also proved the potential for 3,7-cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene) to replace toxic synthetic drugs and act as natural antioxidants.

Abstract 10039: Cardiomyocyte N-myristoylation Plays an Essential Role During the Development of Heart Failure

Background: N-myristoylation is co- and post-translational modifications of proteins, which involve attachment of myristic acid to the N-terminal glycine. Although N-myristoylation plays important roles in physiological activities such as intracellular signaling, the precise regulation and functional relevance of N-myristoylation in the heart remain to be elucidated. Methods and Results: We applied a click chemistry-based approach to determine the global N-myristoylated protein profiling in the cardiomyocyte. Proteomics analysis identified 41 and 34 N-myristoylated proteins in H9c2 myocytes and neonatal rat cardiomyocytes, and 66.1% of them were discovered for the first time at endogenous levels. The levels of N-myristoylation were significantly decreased by the N-myristoyltransferase (NMT) 2 inhibition with siRNA or adenoviral shRNA knockdown, but not by the NMT1 inhibition. In response to angiotensin II (AII), N-myristoylated levels were differentially altered, and MARCKS was identified as the most down-regulated N-myristoylated protein. Wild-type MARCKS was localized to the cellular membrane, while the mutant MARKS with replacement of N-terminal glycine to alanine (G2A), which lacked the capacity of N-myristoylation, exhibited cytosolic distribution. Cardiomyocyte hypertrophy was exacerbated in mutant G2A MARCKS accompanied by increased levels of histone acetylation and phosphorylation of HDAC4 and CaMKII after AII. Gene transfer delivering NMT2 gene specific to the heart using adeno-associated virus 9 significantly improved pressure overload-induced left ventricular systolic dysfunction, cardiac hypertrophy, and myocardial fibrosis, associated with decreased levels of histone acetylation after the transverse aortic constriction in mice ( Fig ). Conclusion: Cardiomyocyte N-myristoylation plays an essential role in developing heart failure, and its modification by NMT2 may be a novel therapeutic strategy for heart failure.

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate’s reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

Pharmacokinetic profile of sarcin and thionin from Aspergillus giganteus and in vitro validation against human fungal pathogen.

Fungal infections are more predominant in agricultural and clinical fields. Aspergillosis caused by Aspergillus fumigatus leads to respiratory failure in patients along with various illnesses. Due to the limitation of antifungal therapy and antifungal drugs, there is an emergence to develop efficient antifungal compounds (AFCs) from natural sources to cure and prevent fungal infections. The present study deals with the investigation of the mechanism of the active compounds from Aspergillus giganteus against aspergillosis. Primarily, the bioavailability and toxicological properties of antifungal proteins such as, sarcin, thionin, chitinase and their derivatives have proved the efficiency of pharmacokinetic properties of selected compounds. Molecular interactions of selected compounds from A. giganteus with the virulence proteins of A. fumigatus (UDP-N-acetylglucosamine pyrophosphorylase, N-myristoyl transferase and Chitinase) have exhibited a good glide score and druggable nature of the AFCs. The antagonistic potential of AFCs on the pathogen was confirmed by SEM analysis where the shrunken and damaged spores of AFCs treated pathogen were observed. The integrity of A. fumigatus cell membrane and nuclear membrane treated with AFCs were analyzed by determining the release of cellular materials. The effective concentration of AFCs was found to be 250 µg/ml (P<0.0001). The GC-MS profiling has revealed the volatile bioactive metabolites present in A. giganteus. Further, interaction studies might provide more information on the synergism activity with the non-volatile metabolites which leads to the development of novel drugs for the treatment of aspergillosis.

Novel Hits for N-Myristoyltransferase Inhibition Discovered by Docking-Based Screening.

N-myristoyltransferase (NMT) inhibitors that were initially developed for treatment of parasitic protozoan infections, including sleeping sickness, malaria, and leismaniasis, have also shown great promise as treatment for oncological diseases. The successful transition of NMT inhibitors, which are currently at preclinical to early clinical stages, toward clinical approval and utilization may depend on the development and design of a diverse set of drug molecules with particular selectivity or pharmacological properties. In our study, we report that a common feature in the inhibitory mechanism of NMT is the formation of a salt bridge between a positively charged chemical group of the small molecule and the negatively charged C-terminus of an enzyme. Based on this observation, we designed a virtual screening protocol to identify novel ligands that mimic this mode of interaction. By screening over 1.1 million structures downloaded from the ZINC database, several hits were identified that displayed NMT inhibitory activity. The stability of the inhibitor-NMT complexes was evaluated by molecular dynamics simulations. The ligands from the stable complexes were tested in vitro and some of them appear to be promising leads for further optimization.

Synthesis, antileishmanial activity and molecular modeling of new 1-aryl/alkyl-3-benzoyl/cyclopropanoyl thiourea derivatives.

Due to the lack of effective vaccine(s) against leishmania and also pharmacokinetics issues of current drugs, it is necessary to discover new antileishmanial agents. Within this particular study, a series of novel 1-aryl/alkyl-3-benzoyl/cyclopropanoyl thiourea derivatives were synthesized (yields 69-84%) and evaluated as antileishmanial compounds (1-11). Synthetic derivatives were subjected to in vitro antileishmanial assessment against Leishmania major promastigotes by colorimetric MTT assay. Compounds 3 (IC50 38.54µg/mL), 5 (IC50 84.75µg/mL) and 10 (IC50 70.31µg/mL) exhibited higher activities after 48h but were less potent than amphotericin B (IC50 0.19µg/mL). Antileishmanial activities indicated priority of 5-methyl-4-phenyl thiazole over furyl methyl substituents and 4-phenyl thiazole on thiourea nitrogen. N-myristoyltransferase (NMT) was selected as a validated L. major target for molecular docking studies. In silico results indicated the contribution of hydrophobic, π-stacking and H-bond interactions in binding to target. Most of the synthesized derivatives had lower binding affinities to human NMT (hNMT) than leishmanial enzyme. Docking conformations of top-ranked selective binders (compounds 3 and 5) were subjected to 50ns MD simulations inside L. major HMT (LmNMT) active site. MD trajectories were used to extract RMSD, RMSF, Rg and durability of intramolecular/intermolecular H-bonds of the complex. It was observed that compound 3 escaped from LmNMT binding site during simulation period and no stable complex could be envisaged. Unlike 3, compound 5 attained stable binding conformation with converged stability parameters. Although mechanistic details for antileishmanial effects of synthesized derivatives are to be explored, current results may be implicated in further structure-guided approach toward potent antileishmanial agents.

Exploring actinomycetes natural products to identify potential multi-target inhibitors against Leishmania donovani.

Visceral leishmaniasis (VL) is a neglected tropical disease that mainly affects the poor population of the Indian, African, and South American subcontinent. The increasing resistance to antimonial and miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an anti-leishmanial drug with excellent efficacy and safety profile. In this study, three sequential docking protocols (HTVS, SP, and XP) were performed to screen the secondary metabolites (n = 6519) from the actinomycetes source against five key proteins involved in the metabolic pathway of Leishmania donovani. Those proteins were adenine phosphoribosyltransferase (PDB ID: 1QB7), trypanothione reductase (PDB ID: 2JK6), N-myristoyl transferase (PDB ID: 2WUU), pteridine reductase (PDB ID: 2XOX), and MAP kinase (PDB ID: 4QNY). Although the binding energy of top ligands was predicted using the MM-GBSA module of the Schrödinger suite. SP and XP docking mode resulted in 55 multi-targeted ligands against L donovani. MM-GBSA analysis selected the top 18 ligands with good-binding affinity and the binding-free energy for four proteins, as mentioned earlier, when compared with the miltefosine, paromomycin, and a reference ligand selected for each target. Finally, molecular dynamics simulation, post-MD-binding-free energy (MM-PBSA), and principal component analysis (PCA) proposed three best ligands (Adenosine pentaphosphate, Atetra P, and GDP-4-keto-6-deoxymannose) qualifying the above screening parameters and confirmed as a potential drug candidate to fight against Leishmania donovani parasites.