Enzyme Dihydrofolate Reductase Research Articles

DESIGN, SYNTHESIS, AND EVALUATION OF ANTIBACTERIAL POTENTIAL OF HYDRAZONE-TETHERED PYRAZOLE-THIAZOLE DERIVATIVES

Objective: This study includes the investigation of the antimicrobial potential of a series of compounds designed by hybridization of thiazole, hydrazone and pyrazole systems identified as antimicrobial moieties in the literature. The aim was to filter the designed compounds with drugability parameters, synthesize the selected compounds and test their antibacterial potential in silico and in vitro. Material and Method: The drugability properties of synthesized compounds were determined by online scanners and the potential effects of selected compounds on E. coli and S. aureus strains were determined by disk diffusion method. Also, Autodock 4.2 software was used to determine the inhibitory potential of compounds against the dihydrofolate reductase (DHFR) enzyme. Result and Discussion: In our study, among the newly designed hydrazone-linked pyrazole-thiazole compounds, the compounds determined according to their drugability parameters (17a-c) were synthesized with high efficiency. Among the compounds tested for antibacterial activity, Compound 17c formed a zone diameter of 8 mm against E. coli strain and 9 mm against S. aureus strain at a concentration of 80 μg/ml. Also, compound 17c formed a zone diameter of 7 mm against E. coli strain and 8 mm against S. aureus strain at a concentration of 40 μg/ml. Furthermore, the ADMET profiles of the presented compounds indicate that they may have suitable drugability parameters as potential antibacterial agents.

Recent Cutting-Edge Designing Strategies for Mtb-DHFR Inhibitors as Antitubercular Agents.

Tuberculosis (TB) is an obstinate and infectious disease requiring a relatively longer treatment duration than other bacterial infections. The current treatment regime is prolonged and cumbersome, with adverse effects, often leading to nonadherence. The upsurge in TB's multidrug-resistant and extensively drug-resistant strains with evolved resistance to existing drugs has compounded the problems. The last two decades witnessed unprecedented progress in developing TB drugs with better efficacy and reduced toxicity. Of late, inhibitors targeting the dihydrofolate reductase (DHFR) enzyme were being explored and developed as antitubercular drugs. A plethora of diverse molecular cores, such as pteridines, diamino heterocycles, diamino triazoles, and nontraditional cores, were developed recently as Mtb-DHFR targets. Besides the characteristic binding pockets of Mtb-DHFR, an extended hydrophilic binding pocket was also studied for intermolecular interactions with the designed compounds to assess the enzyme specificity. In this study, prominent DHFR inhibitors developed in the last two decades were reported. Key features of the designed compounds, such as the structural similarities with existing pharmacophores, interactions with binding pockets, enzyme selectivity and specificity, and percentage of inhibition, were evaluated. The authors hope the study will help streamline the pharmacological pipeline of Mtb-DHFR inhibitors and bring the investigators one step closer to success.

Local disorder is associated with enhanced catalysis in an engineered photoswitch.

The A. sativa LOV2 domain is commonly harnessed as a source of light-based regulation in engineered optogenetic switches. In prior work, we used LOV2 to create a light-regulated Dihydrofolate Reductase (DHFR) enzyme and showed that structurally disperse mutations in DHFR were able to tune the allosteric response to light. However, it remained unclear how light allosterically activates DHFR, and how disperse mutations modulate the allosteric effect. A mechanistic understanding of these phenomena would improve our ability to rationally design new light-regulated enzymes. We used a combination of Eyring analysis and CD spectroscopy to quantify the relationship between allostery, catalytic activity, and global thermal stability. We found that the DHFR/LOV2 fusion was marginally stable at physiological temperatures. LOV2 photoactivation simultaneously: (1) thermally destabilized the fusion and (2) lowered the catalytic transition free energy of the lit state relative to the dark state. The energetic effect of light activation on the transition state free energy was composed of two opposing forces: a favorable reduction in the enthalpic transition state barrier offset by an entropic penalty. Allostery-tuning mutations in DHFR acted through this tradeoff, either accentuating the enthalpic benefit or minimizing the entropic penalty but never improving both. Many of the allostery tuning mutations showed a negative correlation between the light induced change in thermal stability and catalytic activity, suggesting an activity-stability tradeoff.

Design, synthesis, in vitro and in silico studies of novel piperidine derived thiosemicarbazones as inhibitors of dihydrofolate reductase.

Dihydrofolate reductase (DHFR), an essential enzyme in folate metabolism, presents a promising target for drug development against various diseases, including cancer and tuberculosis. Herein, we present an integrated approach combining in vitro biochemical assays with in silico molecular docking analysis to evaluate the inhibitory potential of 4-piperidine-based thiosemicarbazones 5(a-s) against DHFR. In our in vitro study, a novel series of 4-piperidine-based thiosemicarbazones 5(a-s) were assessed for their inhibitory activity against DHFR enzyme. The synthesized compounds 5(a-s) exhibited potent inhibition with IC50 values in the range of 13.70 ± 0.25 µM to 47.30 ± 0.86 µM. Among all the derivatives 5p displayed highest inhibitory activity. Simultaneously, in silico analysis were performed and compared with standard drug (Methotrexate) to predict the binding affinity and interaction pattern of synthesized compounds with DHFR active site. SAR analysis was done to elucidate how structural modifications impact compound's biological activity, guiding the rational design of potent and selective drug candidates for targeted diseases. These findings may provide a comprehensive assessment of 4-piperdine-based thiosemicarbazones as DHFR inhibitors and contribute to the development of novel therapeutics targeting DHFR-associated diseases.

Synthesis and in silico studies of some new pyrrolylbenzamide derivatives as antitubercular and antimicrobial agents

Eighteen new pyrrolylbenzamide derivatives (6a-6r) were synthesized by reacting N-(2,5-dimethyl- 1H-pyrrol-1-yl)-4-(2-hydrazineyl-2-oxoethoxy)benzamide (4) with substituted benzoic acids (5a-5r) in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, diisopropyl ethylamine and dimethyl formamide. The produced compounds were subjected to molecular docking studies against the dihydrofolate reductase (DHFR) and InhA mycobacterial enzymes. The compounds were biologically screened for antimycobacterial, and antibacterial activities along with InhA and DHFR enzyme inhibition studies. The compounds were also analyzed for ADMET parameters. The compounds 6j, 6l, 6q, and 6r exhibited good antitubercular activity, and compounds 6j, 6l, 6q, and 6r were effective against the Gram-negative Escherichia coli strain. The compounds 6b, 6f, 6j, 6l, and 6q showed good InhA inhibition and compounds 6b, 6c, 6j, 6l, and 6q were effective against DHFR enzyme. Among the synthesized compounds 6j and 6l exhibited promising antitubercular, antibacterial, and effective InhA and DHFR enzyme inhibition values.. KEYWORDS :Benzamide, Dimethylpyrrole, Antimycobacterial activity, Antibacterial activity.

FolA thyA knockout E. coli as a suitable surrogate model for evaluation of antifolate sensitivity against PfDHFR-TS

A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.

The characteristic structural and functional dynamics of P. falciparum DHFR binding with pyrimidine chemotypes implicate malaria therapy design

Dihydrofolate reductase (DHFR) enzyme regulates de-novo folate synthesis in Plasmodium falciparum, hence a critical malaria drug target. Pyrimethamine inhibits DHFR, but resistance and lack of cross-species activity are key challenges. Therefore, this study is set to investigate more potent nitrogenous heterocyclic compounds against DHFR. Docking and MD simulations were employed to gain insight into the compounds’ binding free energies and conformational stabilities, while cross-species activity was determined using sequence alignment. Among the 500 screened compounds, PMD_01 (−12.2 kcal/mol), PMD_02 (−11.1 kcal/mol), PMD_03 (−10.7 kcal/mol), and PMD_04 (−10.7 kcal/mol) have the highest binding affinities against pfDHFR compared to pyrimethamine (−7.9 kcal/mol). Critical amino acids for binding interactions in the active site of pfDHFR include Leu45, Thr107, Ile164, and Thr170. PMD-bound systems showed higher binding free energy than pyrimethamine, except PMD_03. Hydrogen bonding interactions with Gly159, Ser101, Ser104, Ser160, Ile157, and Lys48 played significant roles in the binding interaction of these compounds as opposed to Asp53 in pyrimethamine. The four systems converged around 1.90 Å RMSD and showed more stability than pyrimethamine-bound and unbound pfDHFR. The pocket's residues across the four plasmodia were highly conserved, but Phe116 and Ile164 replaced Met in P. knowlesi and Tyr in the other species. PMD_01, PMD_02, PMD_03, and PMD_04 showed promising inhibition against pfDHFR and are, thus, potential antimalarial agents against plasmodia DHFR homologues.

Computational investigation of the anticancer potential of Sorghum bicolor and Setaria italica phytochemicals against dihydrofolate reductase (DHFR) enzyme

Breast and prostate cancer holds the position of foremost contributors to mortality. Dietary therapies for accompanied by medication are widely recognized as a potential method to successfully tackle cancer. Millet grains are the most ancient food, a perfect combination of proteins, carbohydrates, fiber, macronutrients, micronutrients, and vitamins. This study aims to examine the anticancer potential of Sorghum bicolor (Sorghum) and Setaria italica (Foxtail) phytochemicals. The 50 phytochemicals of sorghum and foxtail millets were retrieved through a literature survey and docked to the Dihydrofolate reductase (DHFR), an enzyme essential for cell growth and proliferation. The top-scoring phytochemicals were filtered and further investigated with active-site residue interaction, drug-likeness, and pharmacokinetics analysis. The ligand stability with the DHFR was evaluated through density functional theory (DFT) based HOMO and LUMO calculations. The results show that caffeic acid, ferulic acid, hesperetic acid, stigmasterol, Cis-p-Coumaric acid, and luteolinidin attained greater stability within the active site of DHFR. These phytochemicals showed a docking score of − 6.4 kcal/mol, − 6.4 kcal/mol, − 6.1 kcal/mol, − 6.4 kcal/mol, − 5.4 kcal/mol, and − 6.7 kcal/mol with DHFR (PDB ID:1BOZ) and flutamide and capecitabine have docking score of − 7.5 and − 8.1 for 1BOZ and − 7.4 and − 7.1 with DHFR (PDB ID:1OHK) respectively. The dynamic interaction at the molecular level validated the stability of these phytochemicals against both DHFR target proteins along with excellent drug-likeness and pharmacokinetic properties. However, the current findings were proven and validated through in-silico experiments to validate above identified phytochemicals as DHFR inhibitors, so millets are used as therapeutics for breast and prostate cancer.

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Methotrexate Induced Oral Mucositis and Bone Marrow Suppression in Psoriatic Patient

Methotrexate is an antimetabolite which binds to the dihydrofolate reductase enzyme and used in the treatment of malignant disorders and autoimmune diseases. Myelosuppression is a serious complication of methotrexate toxicity. The following case describes a case of 67 year old male patient diagnosed with psoriasis and wrongly taken methotrexate 7.5 mg daily for 3 days and twice daily for 4 days. He presented with oral mucositis and his lab reports showed myelosuppression. Our case emphasizes the significance of effective patient counselling to ensure the understanding of prescription and to prevent drug toxicity. The key learning points of our case report include the importance of adhering to prescribed medication regimens, awareness of MTX toxicity, early initiation of folinic acid as an antidote, vigilant monitoring of toxicity, and effective patient education on proper medication use. Adherence to established MTX therapy guidelines in dermatological conditions is crucial to minimize the risk of patient.

Cymoxanil disrupts RNA synthesis through inhibiting the activity of dihydrofolate reductase.

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

Computational Evidence for the Anticancer Activity of Phytochemical Constituents of Terminalia Chebula: An In‐Silico Attempt

AbstractNatural compounds represent Secondary metabolites shaped and endorsed by nature over countless years, showcasing both unique chemical variety and a corresponding range of bioactivities, along with drug‐like attributes. Plant‐derived remedies are projected to comprise approximately 25 % of pharmaceuticals in developed nations. Terminalia chebula earns its title as the “King of medicines” due to its multifaceted pharmacological properties. The phytochemical elements within Terminalia chebula were assessed for their interaction with the human dihydrofolate reductase enzyme (5HT4.pdb) due to its significant role in the development of colorectal cancer. The American Cancer Society provides updated CRC statistics based on incidence from population‐based cancer registries and mortality from the National Center for Health Statistics. In 2023, approximately 153,020 individuals will be diagnosed with CRC and 52,550 will die from the disease, including 19,550 cases and 3750 deaths in individuals younger than 50 years. The compounds TC2 (−8.61 Kcal/mol), TC15 (−7.02 Kcal/mol), and TC23 (−6.98 Kcal/mol) demonstrated higher glide scores. Additionally, TC2 (−71.21 Kcal/mol), TC12 (−101.63 Kcal/mol), TC15 (−81.40 Kcal/mol), and TC29 (−77.47 Kcal/mol) exhibited stronger binding affinities (ΔG Bind) to the target. Based on the outcomes of both docking and MM‐GBSA analyses, TC2 was selected for further investigation via molecular dynamics (MD) simulations, which illustrated the stability of the TC2/5HT4 complex. Among the top‐ranked compounds, TC2 adhered to Lipinski's guidelines for drug‐likeness without any violations. Inhibiting the human DHFR enzyme indirectly hampers the progression of colon cancer. The findings from this in‐silico investigation serve as a foundational basis for potential in vitro and in vivo research.

Solution Ionic Strength Can Modulate Functional Loop Conformations in E. coli Dihydrofolate Reductase.

The observation of multiple conformations of a functional loop (termed M20) in the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a closed conformation to an occluded conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered. Molecular dynamics simulations of ecDHFR in model CaCl2 solutions of varying molar ionic strengths IM reveal a substantial free energy barrier between occluded and closed M20 loop states at IM exceeding the E. coli threshold (∼0.24 M). This barrier may facilitate crystallization of ecDHFR in the occluded state, consistent with ecDHFR structures obtained at IM exceeding 0.3 M. At lower IM (≤0.15 M), the M20 loop can explore the occluded state, but prefers an open/partially closed conformation, again consistent with ecDHFR structures. Our findings caution against using ecDHFR structures obtained at nonphysiological ionic strengths in interpreting catalytic events or in structure-based drug design.

Design, synthesis, antimicrobial evaluation, and molecular modeling of new sulfamethoxazole and trimethoprim analogs as potential DHPS/DHFR inhibitors

Trimethoprim (TMP) and sulfonamides and are typically utilized to hinder the activities of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively. In this context, we aimed to enhance the understanding of the inhibitory effects on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes by synthesizing and evaluating two series of compounds. These compounds, designed as derivatives of p-aminobenzoic acid (related to sulfamethoxazole, SUL) and trimethoprim analogs, were rigorously tested for their in vitro antimicrobial efficacy and their ability to hinder the activities of DHFR and DHPS enzymes.Among the synthesized compounds, compounds 6 and 10 demonstrated exceptional antimicrobial efficacy against a diverse spectrum of bacterial and fungal strains. The assessment of these compounds also included their impact on biofilm formation, evaluated through crystal violet staining. Our in vitro enzyme assays revealed that compound 10 emerged as the most potent inhibitor of DHPS, with an IC50 value of 224.91 μg/mL, surpassing SUL by 1.6 times. Simultaneously, Compound 6 exhibited particularly potent DHFR-inhibitory action, with an IC50 value of 3.731 μg/mL, surpassing TMP by a factor of three.Molecular modeling studies further elucidated the mode of action, illustrating that compound 10 effectively occupied the DHPS pterin-binding site alongside p-aminobenzoic acid. In contrast, compound 6 was adeptly positioned within the DHFR-binding pocket.In summary, our findings underscore the promising prospects of compounds 6 and 10 in the management of bacterial infections. The results not only demonstrate their remarkable antimicrobial efficacy but also position them as potentially effective in a synergistic combination targeting the DHPS/DHFR enzymatic cascade.

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.

Novel Benzotriazole-β-lactam Derivatives as Antimalarial Agents: Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Many people around the world suffer from malaria, especially in tropical or subtropical regions. While malaria medications have shown success in treating malaria, there is still a problem with resistance to these drugs. Herein, we designed and synthesized some structurally novel benzotriazole-β-lactams using 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid as a key intermediate. To synthesize the target molecules, the ketene-imine cycloaddition reaction was employed. First, The reaction of 1H-benzo[d][1,2,3]triazole with 2-bromoacetic acid in aqueous sodium hydroxide yielded 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid. Then, the treatment of 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid with tosyl chloride, triethyl amine, and Schiff base provided new β-lactams in good to moderate yields.The formation of all cycloadducts was confirmed by elemental analysis, FT-IR, NMR and mass spectral data. Moreover, X-ray crystallography was used to determine the relative stereochemistry of 4a compound. The in vitro antimalarial activity test was conducted for each compound against P. falciparum K1. The IC50 values ranged from 5.56 to 25.65 μM. A cytotoxicity profile of the compounds at 200 μM final concentration revealed suitable selectivity of the compounds for malaria treatment. Furthermore, the docking study was carried out for each compound into the P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site to analyze their possible binding orientation in the active site.

The Journey of Methotrexate and Potential Alternative Pharmacotherapies for Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is a systemic inflammatory auto-immune disorder chiefly described by synovitis followed by extra-articular manifestations of organ association such as pneumonia in addition to the clinical symptoms that cause long-term joint damage starting from the small joints and gradually progressing to the larger ones. Early diagnosis is considered a major improvement for the most desirable outcomes. Methotrexate (MTX), an antifolate, has been the gold standard therapy in use for over forty years as an anchor drug. This drug started out as an anti-cancer drug in large doses and is now in use to treat rheumatoid arthritis at very low doses. The treatments for rheumatoid arthritis aim to curb the swelling in the body and protect the joints from further damage. Recent research has seen an increase in the use of combination therapies with Methotrexate. In this paper, we present a summary of the current drug in use and its side effects, associated with RA. The paper gives an account of alternate modes of treatment that have been explored for the treatment of rheumatoid arthritis. Initially designed to inhibit the enzyme dihydrofolate reductase and treat various types of cancer, methotrexate found application as an anti-rheumatic drug in 1984 although suggestions for the same have been made since the 1950s. Since then, a substantial amount of clinical evidence has been obtained to clearly indicate the cytotoxic activity of the drug against the cells responsible for joint inflammation associated with RA. Thus, methotrexate is a clear choice when it comes to treating RA despite the advent of other lines of treatment being explored and implemented.

Synthesis, in vitro biological evaluation and in silico studies of novel pyrrolidine derived thiosemicarbazones as dihydrofolate reductase inhibitors.

Dihydrofolate reductase (DHFR) is a crucial enzyme involved in folate metabolism and serves as a prime target for anticancer and antimicrobial therapies. In this study, a series of 4-pyrrolidine-based thiosemicarbazones were synthesized and evaluated for their DHFR inhibitory activity. The synthesis involved a multistep procedure starting from readily available starting materials, leading to the formation of diverse thiosemicarbazone 5(a-r) derivatives. These compounds were then subjected to in vitro assays to evaluate their inhibitory potential against DHFR enzyme. The synthesized compounds 5(a-r) exhibited potent inhibition with IC50 values in the range of 12.37 ± 0.48 μM to 54.10 ± 0.72 μM. Among all the derivatives 5d displayed highest inhibitory activity. Furthermore, molecular docking and ADME studies were performed to understand the binding interactions between the synthesized compounds and the active site of DHFR. The in vitro and in silico data were correlated to identify compounds with promising inhibitory activity and favorable binding modes. This comprehensive study provides insights into the structure-activity relationships of 4-pyrrolidine-based thiosemicarbazones as DHFR inhibitors, offering potential candidates for further optimization towards the development of novel therapeutic agents.

Isolation of Lactobacillus and Enzymatic Activity of Dihydrofolate Reductase (DHFR) in Musa spp.

Background: Bananas grown in the tropic and subtropic regions are among the important crops worldwide. Banana is a general term embracing a number of species or hybrids in the genus Musa, family Musceae. Objective: The present research discusses extensively the breakthrough in the utilization of Musa i.e. Banana from its unripe to ripening phase in order to conclude the presence of Lactobacilli and enzymatic activity of Dihydrofolate Reductase (DHFR). DHFR, a ubiquitous enzyme, is involved in the metabolism and stability of folate (vitamin B9), and causes the reduction of dihydrofolate into tetrahydrofolate (folate or vit B9), which is utilized in amino acids and purine production. It has been studied that the deficiency of DHFR or its inhibition can lead to Megaloblastic anemia, Neural tube defects, improper synthesis of DNA and RNA, colorectal cancer, etc. The presence of the DHFR enzyme in bananas can be utilized in the treatment of diseases related to deficiency of folate, which can become a cheaper and cost-effective way to keep a balance in the folate metabolism and keep the activity of the enzyme maximum. Methodology: The fresh unripe banana samples were collected from an urban fruit market in Karachi, serially diluted, and inoculated into the Nutrient agar, Mannitol salt agar, and De Man, Rogosa, Sharpe agar media to check for microbial growth. The enzymatic activity of DHFR and its association with Musa was also checked by using DHFR substrate. Results: The results concluded from our experiment are that the Musa contains Gram-positive bacilli that were further tested to characterize the microorganism into its respective genus, indicating the presence of Lactobacillus in the banana. The enzymatic activity of DHFR was observed at maximum on day 2 and minimum enzymatic activity was observed on day 4. Conclusion: Hence, people suffering from folic acid deficiency, DHFR enzyme and consumption of day 2 bananas can be prescribed. Not only this, the presence of Lactobacilli as probiotics in bananas restore consumer’s intestinal microbial dysbiosis and maintain healthy intestinal ecosystem. Furthermore, herbal medicines can be made from banana and given to patients in order to restore the deficiency.

Pyrimidine derivatives: Their significance in the battle against malaria, cancer and viral infections

Pyrimidine derivatives play a significant role in combating viral infections, malaria, and cancer due to their diverse pharmacological properties. Pyrimidine derivatives, particularly nucleoside analogs, have been widely used as antiviral agents. They interfere with viral replication by inhibiting DNA or RNA synthesis.Examples include drugs like acyclovir (used against herpes viruses), lamivudine (for HIV and hepatitis B), and remdesivir (for COVID-19). These compounds help to manage and treat various viral infections, reducing their severity and spread. Pyrimidine derivatives are key components of antimalarial drugs like pyrimethamine and proguanil. They target the parasite Plasmodium falciparum's dihydrofolate reductase enzyme, essential for its survival. These drugs have been instrumental in combating malaria, a major global health concern, by inhibiting the growth of the malaria parasite within the human body. Pyrimidine analogs are used in chemotherapy to target rapidly dividing cancer cells. Drugs like 5-fluorouracil (5-FU) and cytarabine inhibit DNA synthesis in cancer cells, leading to cell death. These derivatives have been crucial in the treatment of various cancers, helping to slow down tumor growth and improve patient outcomes. Overall, this article aims to consolidate existing knowledge on the topic, synthesize relevant information, and contribute to a better understanding of the potential applications of pyrimidine derivatives in the fields of oncology, virology, and tropical medicine.