Dihydrofolate Reductase Inhibitors Research Articles

Identification of novel peptide inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR): molecular docking and MD simulation studies

The presence of drug-resistant variants of Plasmodium parasites within the population has presented a substantial obstacle to the eradication of Malaria. As a result, numerous research groups have directed their efforts towards creating new medication candidates that specifically target parasites. In this study, our main objective was to identify tri-peptide inhibitors for Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) with the aim of finding a new peptide that exhibits superior binding properties compared to the current inhibitor, WR99210. In order to achieve this objective, a virtual library consisting of 8000 tripeptides was generated and subjected to computational screening against wild-type PfDHFR. The purpose of this screening was to discover the most effective binders at the active site. The four most optimal tripeptides identified (Trp-Trp-Glu, Trp-Phe-Tyr, Phe-Trp-Trp, Tyr-Trp-Trp) exhibited significant non-covalent interactions inside the active site of PfDHFR and had binding energies ranging from −9.5 to −9.0 kcal/mol and WR99210 had a binding energy of −6.2 kcal/mol. A 250 ns Molecular Dynamics (MD) simulation was performed to investigate the kinetic and thermodynamic characteristics of the protein-ligand complexes. The Root Mean Square Deviation (RMSD) values for the optimal tripeptides fell within the allowed range, indicating the stability of the ligands inside the protein complex. The Ki value for the most effective tripeptide was 0.3482 µM, whereas WR99210 had a Ki value of 1.02 µM. This article presents the initial discovery of peptide inhibitors targeting PfDHFR. In this text, we provide a comprehensive explanation of the interactions that occur between peptides and the enzyme. Communicated by Ramaswamy H. Sarma

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Abstract LB404: ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. RMS tumors are classified into four subtypes: embryonal, alveolar, spindle cell/sclerosing, and pleomorphic. Treatment options for RMS include a combination of surgery, chemotherapy, and radiation therapy and regimens for patients with relapsed and metastatic RMS remain limited. However, a recent study has reported that pharmacological inhibitors targeting ataxia telangiectasia and Rad3-related protein (ATR) are currently undergoing phase I/II trials and may be utilized for the treatment of alveolar RMS. Interestingly, we identified that the gene signature of replication stress positively correlated with dihydrofolate reductase (DHFR) expression in the cancer cell line database (CellMinerCDB, discover.nci.nih.gov/cellminercdb; Pearson correlation (r)=0.66). DHFR is an important therapeutic target for cancer and autoimmune diseases, and its inhibitors are routinely used and clinically approved. DHFR inhibitors are known to exhibit their anticancer effects by inhibiting nucleotide synthesis and DNA replication. In this study, we aimed to explore a potential synergistic interaction using a combination treatment of ATR inhibitors (elimusertib and ceralasertib) and DHFR inhibitors (pralatrexate, methotrexate, and raltitrexated) in multiple RMS cell lines. We used a drug matrix reduction format and found that combined treatment of ATR inhibitors and DHFR inhibitors induced a synergistically decreased RMS cell viability, showing higher synergy in fusion-positive RMS cells than in fusion-negative RMS cells. Mechanistically, ATR inhibitors abrogated DNA damage response checkpoint CHK1 activation caused by DHFR inhibitor, leading to increased γH2AX and decreased DNA replication activity. Accordingly, the accumulation of DNA damage in combination with ATR inhibitor and DHFR inhibitor triggered higher cell cycle arrest and apoptotic cell death than single agent treatment of DHFR inhibitor. Our findings warrant further research into identifying genomic regions where the DNA damage is enriched in combination treatment by using END-sequencing and synthesis-associated with repair sequencing (SAR-seq). In conclusion, this study provides promising rationale for the combination treatment of ATR and DHFR inhibitors to treat RMS patients, including those with advanced, relapsed, and metastatic states. Citation Format: Ukhyun Jo, George K Annor, Jack F. Shern. ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB404.

Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica.

In Salmonella enterica, the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an alr ridA strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the alr ridA strain were identified as lesions in folE, which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a folE lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.

Design, Synthesis, and Biological Evaluation of 5-(5-Iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2,4-diamine (AF-353) Derivatives as Novel DHFR Inhibitors against Staphylococcus aureus.

The high lethality of Staphylococcus aureus infections and the emergence of antibiotic resistance make the development of new antibiotics urgent. Our previous work identified a hit compound h1 (AF-353) as a novel Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor. Herein, we analyzed the antimicrobial profile of h1 and performed a comprehensive structure-activity relationship (SAR) assay based on h1. The representative compound j9 exhibited potent antibacterial activity against S. aureus without cross-resistance to other antimicrobial classes. Multiple genetic and biochemical approaches showed that j9 directly binds to SaDHFR, resulting in strong inhibition of its enzymatic activity (IC50 = 0.97 nM). Additionally, j9 had an acceptable in vivo safety profile and oral bioavailability (F = 40.7%) and also showed favorable efficacy in a mouse model of methicillin-resistant S. aureus (MRSA) skin infection. Collectively, these findings identified j9 as a novel SaDHFR inhibitor with the potential to combat drug-resistant S. aureus infections.

Molecular Docking, In silico ADMET Study and Synthesis of Quinoline Derivatives as Dihydrofolate Reductase (DHFR) Inhibitors: A Solvent-free One-pot Green Approach Through Sonochemistry

Background: Quinoline derivatives have evinced their biological importance in targeting bacteria by inhibiting Dihydrofolate reductase. H2SO4 was successfully applied as an acid catalyst for a green, efficient, and one-pot solvent-free synthesis of quinoline derivatives using sonochemistry approach from various aromatic amines and glycerol with affording yield up to 96% within 6-10 min. Objective: In this study, the synthesis, characterization, and biological assessment of fifteen quinoline derivatives (1-15) as potential DHFR inhibitors were carried out. The target compounds were docked to study the molecular interactions and binding affinities with the 1DLS enzyme. Methods: The synthesized molecules were characterized using IR, MASS, and 1H and 13C NMR. The Insilico molecular docking study was carried out through target Human Dihydrofolate Reductase (DHFR) retrieved from a protein data bank having PDB ID: 1DLS and the antimicrobial activity of all synthesized compounds were tested against Human Dihydrofolate Reductase(DHFR) enzyme by using in-vitro DHFR assay kit. Results: The molecular docking results revealed that compounds 2 and 6 have the lowest binding energy and good binding affinity with the DHFR enzyme. In-silico ADMET predictions revealed that all bestscored compounds had good absorption and drug-like properties for potential use as DHFR inhibitors to treat bacterial infection. The in vitro studies revealed that compounds 2 and 6 show potent DFHR inhibitory activity against gram-positive and gram-negative with IC50 = 12.05 ± 1.55 μM and 10.04 ± 0.73 μM, respectively. While compounds 12, 13, and 15 exhibited moderate antimicrobial activity through DHFR inhibition with IC50= 16.33 ± 0.73 μM, 17.02 ± 1.55 μM, and 18.04 ± 1.05 μM, respectively. Conclusion: This environmentally benign sonochemistry-based approach for synthesizing quinoline derivatives could be affordable for large-scale production and become a potential lead candidate for developing a new quinoline-based antimicrobial agent.

Quassinoids from Eurycoma longifolia as Potential Dihydrofolate Reductase Inhibitors: A Computational Study.

Quassinoids are degraded triterpene compounds that can be obtained from various species of the Simaroubaceae plant family, including Eurycoma longifolia. Quassinoids are the major compounds in E. longifolia, and they are known to have various medicinal potentials, such as anticancer and antimalarial properties. Dihydrofolate reductase (DHFR) was reported to be one of the important targets for certain anticancer and antimalarial drugs. Twelve quassinoids from E. longifolia were identified to have anticancer effects based on their IC50 values. This study aimed to evaluate the interactions of these twelve quassinoids with DHFR via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Twelve quassinoids from E. longifolia and their interactions with DHFR were evaluated via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Their drug-likeness and pharmacokinetic properties were also assessed using the ADMETlab 2.0 program. The molecular docking results showed that eleven quassinoids showed better docking scores than methotrexate, in which the binding energy (BE) of these quassinoids ranged from - 7.87 to -9.58 kcal/mol. Their inhibition constant (Ki) ranged from 0.095 to 1.71 μM. At the same time, the BE and Ki values for methotrexate were -7.80 kcal/mol and 1.64 μM, respectively. From the analysis, 6-dehydrolongilactone and eurycomalide B are among the twelve compounds that showed great potential as hit-to-lead compounds based on the docking score on DHFR, drug-likeness, and ADMET properties. These results suggest a great potential to pursue validation studies via in vitro and in vivo models.

Maternal exposure to folate antagonists and susceptibility to congenital heart disease in offspring: A systematic review and meta-analysis.

The objective of this meta-analysis was to determine whether maternal exposure to folate antagonists is associated with increased rates of congenital heart disease in offspring. A comprehensive search for articles in the MEDLINE (PubMed) and EMBASE databases published up to 21 August 2023 was performed. The search strategy was not limited by study design but only for articles in the English language. Analysis of 6 cohort studies and 5 cross-sectional studies, published between 1976 and 2020, showed significant increase in rate of congenital heart disease (odds ratio 1.55, 95% confidence interval, 1.28-1.87) when exposed to folate antagonists compared with the control. Further subgroup analysis showed the increased rate for exposure to both dihydrofolate reductase inhibitors and antiepileptic drugs separately. No differences were observed when analyses were stratified by timing of study. Administration of folate antagonists within the 12-week period preceding conception and throughout the second and third months of gestation exhibited a statistically significant elevation in the susceptibility to congenital heart diseases. Notably, the protective effect of folic acid supplementation was reported in cases of congenital heart disease linked to dihydrofolate reductase inhibitors but not that associated with antiepileptic drugs.

Repurposing FDA-approved drugs to target malaria through inhibition of dihydrofolate reductase in the folate biosynthesis pathway: A prospective approach.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that regulates the biosynthesis of tetrahydrofolate among various species of Plasmodium parasite. It is a validated target of the antifolate drug pyrimethamine (Pyr) in Plasmodium falciparum (Pf), but its clinical efficacy has been hampered due to the emergence of drug resistance. This has made the attempt to screen Food & Drug Administration-approved drugs against wild- and mutantPfDHFR by employing an in-silico pipeline to identify potent candidates. The current study has followed a virtual screening approach for identifying potential DHFR inhibitors from DrugBank database, based on a structure similarity search of candidates, followed by absorption, distribution, metabolism, and excretion estimation. The screened drugs were subjected to various parameters like docking, molecular mechanics with generalized born and surface area solvation calculations, and molecular simulations. We have thus identified twopotential drug candidates, duloxetine and guanethidine, which can be repurposed to be tested for their efficacy against wild type and drug resistant falciparum malaria.

A Comprehensive Review of Toxoplasmosis: Serious Threat to Human Health

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. Despite infecting a major fraction of the global population, T. gondii rarely results in clinically significant disease. Cats are the only known definitive host for this parasite, which sheds millions of oocysts in its feces every day, which then sporulate and become infective in the environment. This comprehensive review article aims to explain the etiology, pathogenesis, epidemiology, transmission, clinical symptoms, diagnosis, risk factors, public health importance, economic effect, treatment, and prevention of toxoplasmosis. A search for various publications in English with the criteria of reviewing articles explaining toxoplasmosis was carried out. T. gondii reproduces through two life cycles, namely the sexual cycle and the asexual cycle. In general, consuming parasite cysts in tainted food or water is how humans and other warm-blooded animals become infected with T. gondii. Nearly every region of the world has reported incidences of toxoplasmosis in humans, and around one-third of people are susceptible to latent infection. According to the reports, the main ways through which diseases spread are by water, tainted food, eating tissue cysts or oocysts, and congenital transmission. Infected individuals may experience asymptomatic cervical lymphadenopathy during an acute systemic infection. Diagnostic evaluation is very important for early detection, prevention of transmission, and as a reference for treatment options for infected pregnant women. Consuming undercooked meat is traditionally seen as a significant risk factor for developing toxoplasmosis. The impact of toxoplasmosis is very significant in humans because it causes abortion and disease in newborns, resulting in serious economic losses. To treat toxoplasmosis, dihydropteroate synthetase and dihydrofolate reductase inhibitors are advised. Toxoplasma transmission to humans can be avoided by thoroughly washing your hands with soap after handling meat, poultry, or shellfish.

DihydrofolateReductase (DHFR) Inhibitors: A Comprehensive Review.

Dihydrofolate reductase (DHFR) is an indispensable enzyme required for the survival of most prokaryotic and eukaryotic cells as it is involved in the biosynthesis of essential cellular components. DHFR has attracted a lot of attention as a molecular target for various diseases like cancer, bacterial infection, malaria, tuberculosis, dental caries, trypanosomiasis, leishmaniasis, fungal infection, influenza, Buruli ulcer, and respiratory illness. Various teams of researchers have reported different DHFR inhibitors to explore their therapeutic efficacy. Despite all the progress made, there is a strong need to find more novel leading structures, which may be used as better and safe DHFR inhibitors, especially against the microorganisms which are resistant to the developed drug candidates. This review aims to pay attention to recent development, particularly made in the past two decades and published in this field, and pay particular attention to promising DHFR inhibitors. Hence, an attempt has been made in this article to highlight the structure of dihydrofolate reductase, the mechanism of action of DHFR inhibitors, most recently reported DHFR inhibitors, diverse pharmacological applications of DHFR inhibitors, reported in-silico study data and recent patents based on DHFR inhibitors to comprehensively portray the current scenery for researchers interested in designing novel DHFR inhibitors. A critical review of recent studies revealed that most novel DHFR inhibitor compounds either synthetically or naturally derived are characterized by the presence of heterocyclic moieties in their structure. Non-classical antifolates like trimethoprim, pyrimethamine, and proguanil are considered excellent templates to design novel DHFR inhibitors, and most of them have substituted 2,4-diamino pyrimidine motifs. Targeting DHFR has massive potential to be investigated for newer therapeutic possibilities to treat various diseases of clinical importance.

Preclinical translational platform of neuroinflammatory disease biology relevant to neurodegenerative disease

Neuroinflammation is a key driver of neurodegenerative disease, however the tools available to model this disease biology at the systems level are lacking. We describe a translational drug discovery platform based on organotypic culture of murine cortical brain slices that recapitulate disease-relevant neuroinflammatory biology. After an acute injury response, the brain slices assume a chronic neuroinflammatory state marked by transcriptomic profiles indicative of activation of microglia and astrocytes and loss of neuronal function. Microglia are necessary for manifestation of this neuroinflammation, as depletion of microglia prior to isolation of the brain slices prevents both activation of astrocytes and robust loss of synaptic function genes. The transcriptomic pattern of neuroinflammation in the mouse platform is present in published datasets derived from patients with amyotrophic lateral sclerosis, Huntington’s disease, and frontotemporal dementia. Pharmacological utility of the platform was validated by demonstrating reversal of microglial activation and the overall transcriptomic signature with transforming growth factor-β. Additional anti-inflammatory targets were screened and inhibitors of glucocorticoid receptors, COX-2, dihydrofolate reductase, and NLRP3 inflammasome all failed to reverse the neuroinflammatory signature. Bioinformatics analysis of the neuroinflammatory signature identified protein tyrosine phosphatase non-receptor type 11 (PTPN11/SHP2) as a potential target. Three structurally distinct inhibitors of PTPN11 (RMC-4550, TN0155, IACS-13909) reversed the neuroinflammatory disease signature. Collectively, these results highlight the utility of this novel neuroinflammatory platform for facilitating identification and validation of targets for neuroinflammatory neurodegenerative disease drug discovery.

Methotrexate-induced Hyperpigmentation in a Patient with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic progressive systemic and inflammatory disease affecting the lining of the synovial membranes, leading to inflammatory polyarthritis and extraarticular involvement in a symmetrical way. RA is known to be an autoimmune condition, which means that the body’s natural self-defense system gets confused and starts to attack the body’s healthy tissues. Treatment of RA is usually with disease-modifying antirheumatic drugs (DMARDs), but if left untreated, the risk of morbidity, mortality rate, and socioeconomic burdens will increase. Methotrexate (MTX) is one of the folic acid antagonists and one of the conventional synthetic DMARDs. It is the most common drug for the treatment of RA. MTX is a dihydrofolate reductase inhibitor that decreases purine metabolism, which affects cell division. MTX can also cause different types of cutaneous side effects, such as acral erythema, urticaria, ulcerations of the oral mucosa, burning sensation of the skin, multiform erythema, vasculitis, and hyperpigmentation to sun-exposed area. In this case report, we describe a 31-year-old woman who had RA and then developed MTX-induced hyperpigmentation.

In-silico studies of hydroxyxanthone derivatives as potential pfDHFR and pfDHODH inhibitor by molecular docking, molecular dynamics simulation, MM-PBSA calculation and pharmacokinetics prediction

The investigation of hydroxyxanthone derivatives has been conducted, including molecular docking, molecular dynamics simulation MM-PBSA binding energy calculation, and pharmacokinetics prediction of the potential plasmodium falciparum dihydrofolate reductase (pfDHFR) and plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) inhibitor. The Docking result showed that compound 1,3,6,7-tetrahydroxy-5,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X16) was found to be the best ligand with good inhibitory action against pfDHFR. Meanwhile, the pfDHODH protein was compounded 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14). Additionally, the hydroxyxanthone X16 complex showed more excellent stability in the molecular dynamics simulation of the pfDHFR protein than the ligand WR99210 and chloroquine. The MM-PBSA calculation showed that compound X16 had lower binding energy than ligand WR99210. However, 1,3-dihydroxy-8-(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X4), 1,3,6,7-tetrahydroxy-8-nitro-9H-xanthen-9-one (X10), 1,3,6,7-tetrahydroxy-9-oxo-9H-xanthene-8-sulfonic acid (X11), and 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14) complexes were shown to be more stable than chloroquine and to have the same stability when compared to the native ligand A26, according to a molecular dynamics simulation conducted in pfDHODH protein. The MM-PBSA calculation showed that compound X14 had lower binding energy than ligand A26. The hydroxyxanthones X4, X10–11, X14, and X16 fulfill Lipinski's rule parameters in terms of physicochemical and ADMET qualities and parameters related to absorption, distribution, metabolism, excretion, and toxicity tests. To sum up, hydroxyxanthones X4, X10–11, X14, and X16 have the potential to be antimalarial medications, but more in vivo and in vitro testing is needed to confirm this.

In-silico evaluation of Bismurrayaquinone-A phytochemical as a potential multifunctional inhibitor targeting dihydrofolate reductase: implications for anticancer and antibacterial drug development

Dihydrofolate reductase (DHFR) has gained significant attention in drug development, primarily due to marked distinctions in its active site among different species. DHFR plays a crucial role in both DNA and amino acid metabolism by facilitating the transfer of monocarbon residues through tetrahydrofolate, which is vital for nucleotide and amino acid synthesis. This considers its potential as a promising target for therapeutic interventions. In this study, our focus was on conducting a virtual screening of phytoconstituents from the IMPPAT2.0 database to identify potential inhibitors of DHFR. The initial criterion involved assessing the binding energy of molecules against DHFR and we screened top 20 compounds ranging energy −13.5 to −11.4 (kcal/Mol) while Pemetrexed disodium bound with less energy −10.2 (kcal/Mol), followed by an analysis of their interactions to identify more effective hits. We prioritized IMPHY007679 (Bismurrayaquinone-A), which displayed a high binding affinity and crucial interaction with DHFR. We also evaluated the drug-like properties and biological activity of IMPHY007679. Furthermore, MD simulation was done, RMSD, RMSF, Rg, SASA, PCA and FEL explore the time-evolution impact of IMPHY007679 comparing it with a reference drug, Pemetrexed disodium. Collectively, our findings suggest that IMPHY007679 recommend further investigation in both in vitro and in vivo settings for its potential in developing anticancer and antibacterial therapies. This compound holds promise as a valuable candidate for advancing drug research and treatment strategies. Communicated by Ramaswamy H. Sarma

Discovery of Novel Rhizoctonia solani DHFR Inhibitors as Fungicides Using Virtual Screening.

Dihydrofolate reductase (DHFR) is an essential enzyme in the folate pathway and has been recognized as a well-known target for antibacterial and antifungal drugs. We discovered eight compounds from the ZINC database using virtual screening to inhibit Rhizoctonia solani (R. solani), a fungal pathogen in crops. These compounds were evaluated with in vitro assays for enzymatic and antifungal activity. Among these, compound Hit8 is the most active R. solani DHFR inhibitor, with the IC50 of 10.2 μM. The selectivity of inhibition is 22.3 against human DHFR with the IC50 of 227.7 μM. Moreover, Hit8 has higher antifungal activity against R. solani (EC50 of 38.2 mg L-1) compared with validamycin A (EC50 of 67.6 mg L-1), a well-documented fungicide. These results suggest that Hit8 may be a potential fungicide. Our study exemplifies a computer-aided method to discover novel inhibitors that could target plant pathogenic fungi.

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus.

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.

“Design, Synthesis, and Molecular Docking Analysis of Novel Benzo[4,5]imidazo[1,2-a]pyrimidine Hybrids Targeting Plasmodium falciparum DHFR: A Comprehensive In Silico and Biological Study”

Synthesis of Benzo[4,5]imidazo[1,2-a]pyrimidine derivatives using an environmentally friendly Biginelli-type reaction in ionic liquid DIPEAc was performed. Remarkably, this ionic liquid exhibits efficient recyclability over five cycles. Synthesized compounds underwent rigorous in vitro antimalarial efficacy screening, complemented by computational and in vitro studies, assessing their potential as Plasmodium falciparum dihydrofolate reductase (Pf-DHFR) inhibitors. A robust 3D-QSAR model was established and validated, elucidating structure-activity relationships. Estimated ADMET descriptors highlighted favorable pharmacokinetics, suggesting their role in new antimalarial drug development. This study exemplifies the synergy of sustainable synthesis, enzyme inhibition, and pharmacokinetic profiling, promising a transformative outlook for antimalarial innovation.

New Pyrazole-Clubbed Pyrimidine or Pyrazoline Hybrids as Anti-Methicillin-Resistant Staphylococcus aureus Agents: Design, Synthesis, In Vitro and In Vivo Evaluation, and Molecular Modeling Simulation.

Two hybrid series of pyrazole-clubbed pyrimidines 5a-c and pyrazole-clubbed pyrazoline compounds 6a,b and 7 were designed as attractive scaffolds to be investigated in vitro and in vivo for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. From the results of the in vitro antibacterial screening, compound 5c showed excellent activity (minimal inhibitory concentration, MIC = 521 μM) when compared with that of the reference antibiotic levofloxacin (MIC = 346 μM). The inhibition of the target dihydrofolate reductase (DHFR) enzyme by compounds 4 and 5a-c (IC50 = 5.00 ± 0.23, 4.20 ± 0.20, 4.10 ± 0.19, and 4.00 ± 0.18 μM, respectively) was found to be better than the reference drug trimethoprim (IC50 = 5.54 ± 0.28 μM). Molecular modeling simulation results have justified the order of activity of all the newly synthesized compounds as DHFR enzyme inhibitors, and compound 5c exhibited the best binding profile (-13.6169386 kcal/mol). Hence, the most potent inhibitor of the DHFR enzyme, 5c, was chosen to be evaluated in vivo for its activity in treating MRSA-induced keratitis in rats and that, in turn, significantly (P < 0.0001) reduced infection in rats when compared to MRSA-treated group results.