Dihydrofolate Reductase Inhibitors Research Articles

Apparent Absence of Selective Pressure on Pneumocystis jirovecii Organisms in Patients with Prior Methotrexate Exposure.

Pneumocystis jirovecii infections occur in patients treated with methotrexate (MTX) because of immunosuppressive effects of this highly potent dihydrofolate reductase (DHFR) inhibitor. Conversely, MTX may act as an anti-P. jirovecii drug and consequently may exert a selective pressure on this fungus. In this context, we compared the sequences of the dhfr gene of P. jirovecii isolates obtained from two groups of patients with P. jirovecii infections. The first group, with systemic diseases or malignancies, had prior exposure to MTX (21 patients), whereas the second group (22 patients), the control group, did not. Three single nucleotide polymorphisms (SNPs) were observed at positions 278, 312, and 381. The first one was located in the intronic region and the two others were synonymous. Based on these SNPs, three P. jirovecii dhfr alleles, named A, B, and C, were specified. Allele A was the most frequent, as it was observed in 18 patients (85.7%) and in 16 patients (72.7%) of the first and second groups, respectively. No significant difference in P. jirovecii dhfr gene diversity in the two patient groups was observed. In conclusion, these original results suggest that MTX does not exert an overt selective pressure on P. jirovecii organisms.

Insights on attenuating autophagy cellular and molecular pathways versus methotrexate-induced toxicity via liposomal turmeric therapy

BackgroundMethotrexate (MX), a competitive inhibitor of dihydrofolate reductase, can inhibit DNA and RNA production and is a powerful anticancer agent widely utilized in clinical practice for treating nonneoplastic maladies, as psoriasis and rheumatoid arthritis; meanwhile, its probable prescription dose and interval of administration are strictly limited due to dose-related organ damage. Former studies verified that kidney, brain, liver, and lung harms are prospective obstacles of methotrexate administration. To understand the machinery of methotrexate-prompt toxicity, various mechanisms were investigated. The former is an autophagy defense mechanism; autophagy is a self-digesting mechanism responsible for the removal of damaged organelles and malformed proteins by lysosome. The contemporary article hypothesized that turmeric or its liposomal analog could defeat autophagy of MX-induced acute toxicity. Methotrexate, in a dose of 1.5 mg/kg, was administered intravenously followed by turmeric and liposomal turmeric treatment in a dose of 5 mg/kg for 30 days in rats. ResultsIncrement in autophagy (AUTP) consent by MX administration was attenuated by concurrent treatment via turmeric and liposomal turmeric that was reliable on the alteration in apoptotic markers. The assembly of FOXO-3 in serum post methotrexate administration was suppressed by concurrent treatment via liposomal turmeric. Apoptosis/autophagic marker investigation was evaluated through the gene expression of Bax (BCL2-associated X protein)/Bcl2 (B-cell lymphoma 2)/P53 (tumor protein P53)/SiRT-1 (sirtuin silent mating-type information regulation 2 homolog 1) and FOXO-3 (forkhead box transcription factor-3)/ERDJ-4 (endoplasmic reticulum localized DnaJ homologs)/BNP (brain natriuretic peptide B) signaling. The cell death of all cells was categorized to achieve autophagy. Interestingly, Bax/Bcl2/P53/SiRT-1 signaling pathways were downregulated, contributing to inhibiting the initiation of autophagy. Meanwhile, FOXO-3/BNP/ERDJ-4 reduction-implicated noncanonical autophagy pathways were involved in methotrexate-induced autophagy, whereas this change was suppressed when turmeric was administered in liposomal form. ConclusionThese outcomes recommended that liposomal turmeric prevents MX-induced acute toxicity through its autophagy, antioxidant, and antiapoptotic properties.

The Protective Effects of Nutraceutical Components in Methotrexate-Induced Toxicity Models-An Overview.

There are multiple concerns associated with methotrexate (MTX), widely recognized for anti-neoplastic and anti-inflammatory effects in life-threatening disease conditions, i.e., acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, psoriasis, and rheumatoid arthritis, due to long-term side effects and associated toxicity, which limits its valuable potential. MTX acts as an inhibitor of dihydrofolate reductase, leading to suppression of purine and pyrimidine synthesis in high metabolic and turnover cells, targeting cancer and dysregulated immune cells. Due to low discrimination between neoplastic cells and naturally high turnover cells, MTX is prone to inhibiting the division of all fast-dividing cells, causing toxicity in multiple organs. Nutraceutical compounds are plant-based or food-derived compounds, used for their preventive and therapeutic role, ascertained in multiple organ dysfunctions, including cardiovascular disease, ischemic stroke, cancer, and neurodegenerative diseases. Gut microbiota and microbiota-derived metabolites take part in multiple physiological processes, their dysregulation being involved in disease pathogenesis. Modulation of gut microbiota by using nutraceutical compounds represents a promising therapeutic direction to restore intestinal dysfunction associated with MTX treatment. In this review, we address the main organ dysfunctions induced by MTX treatment, and modulations of them by using nutraceutical compounds. Moreover, we revealed the protective mechanisms of nutraceuticals in MTX-induced intestinal dysfunctions by modulation of gut microbiota.

New quinazolinone-based derivatives as DHFR/EGFR-TK inhibitors: Synthesis, molecular modeling simulations, and anticancer activity.

New 2-mercapto-quinazolin-4-one analogs were synthesized and tested for their in vitro anticancer activity, dihydrofolate reductase (DHFR) inhibition, and epidermal growth factor tyrosine kinase (EGFR-TK) inhibition activities. Compound 24, which is characterized by a 2-benzyl-thio function, showed broad-spectrum anticancer activity with high safety profile and selectivity index. The concentrations of 24 causing 50% growth inhibition (GI50 ) and total cell growth inhibition (TGI) and its lethal concentration 50 (LC50 ) were 15.1, 52.5, and 91.2 µM, respectively, using 5-fluorouracil as a positive control. Also, it showed EGFR-TK inhibitory activity with IC50 = 13.40 nM compared to gefitinib (IC50 = 18.14 nM) and DHFR inhibitory potency with 0.30 μM compared to methotrexate (MTX; IC50 = 0.08 μM). In addition, compound 24 caused cell cycle arrest and apoptosis on COLO-205 colon cancer cells. Compounds 37, 21, and 54 showed remarkable DHFR inhibitory activity with IC50 values of 0.03, 0.08, and 0.08 μM, respectively. The inhibitory properties of these compounds are due to an electron-withdrawing group on the quinazolinone ring, except for compound 54. In a molecular modeling study, compound 24 showed the same binding mode as gefitinib as it interacted with the amino acid Lys745 via π-π interaction. Compound 37 showed a similar binding mode as MTX through the binding interaction with Lys68, Asn64 via hydrogen bond acceptor, and Phe31 via arene-arene interaction. The obtained model and substitution pattern could be used for further development.

Human dihydrofolate reductase inhibition effect of 1-Phenylpyrazolo[3,4–d]pyrimidines: Synthesis, antitumor evaluation and molecular modeling study

A new series of pyrazolo[3,4–d]pyrimidine analogues bearing different amino acid conjugates 10a-m were synthesized with the aim to evaluate their antitumor effect through simultaneous inhibition of human dihydrofolate reductase (hDHFR). All novel compounds were tested to screen their enzyme inhibition activity against (hDHFR) beside their in vitro cytotoxicity against six human MTX resistant cancer cell lines namely, human prostate cancer (PC-3), pancreatic human cancer cell lines (BxPC-3), colorectal carcinoma (HCT-116), human hepatocellular carcinoma (HepG-2), cervical carcinoma (HeLa), and mammary gland breast cancer (MCF-7), besides normal immortalized pancreatic cell line (HPDE). Compounds 10e, 10f, 10g inhibited DHFR at considerable low (IC50 < 1 µM) in comparison to MTX (IC50 = 5.61 µM) beside their characteristic cytotoxic effects on different resistant cancer cell lines. Flow cytometry was done for the most active candidate compound 10e against MCF-7 breast cancer cell line. The results illustrated that compound 10e induced apoptosis and arrested MCF-7 cell cycle in the G1/S phase. Western blot for visualization and quantification was used to confirm the capability of compound 10e to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to reduce the expression of antiapoptotic Bcl-2 protein. Molecular modeling studies demonstrated that compound 10e elucidated binding energy of (S= − 8.4390 Kcal/mol) that exceed that of the normal ligand MTX (S= − 8.3951Kcal/mol) in addition to several favorable binding interactions with the active site residues.

Synthesis and Characterization of Functionalized Amino Dihydropyrimidines Toward the Analysis of their Antibacterial Structure-Activity Relationships and Mechanism of Action.

Antibiotic resistance among bacteria puts immense strain on public health. The discovery of new antibiotics that work through unique mechanisms is one important pillar toward combating this threat of resistance. A functionalized amino dihydropyrimidine was reported to exhibit antibacterial activity via the inhibition of dihydrofolate reductase, an underexploited antibacterial target. Despite this promise, little is known about its structure–activity relationships (SAR) and mechanism of activity. Toward this goal, the aza-Biginelli reaction was optimized to allow for the preparation of focused libraries of functionalized amino dihydropyridines, which in some cases required the use of variable temperature NMR analysis for the conclusive assignment of compound identity and purity. Antibacterial activity was examined using microdilution assays, and compound interactions with dihydrofolate reductase were assessed using antimicrobial synergy studies alongside in vitro enzyme kinetics, differential scanning fluorimetry, and protein crystallography. Clear antibacterial SAR trends were unveiled (MIC values from >64 to 4 μg/mL), indicating that this compound class has promise for future development as an antibacterial agent. Despite this, the in vitro biochemical and biophysical studies performed alongside the synergy assays call the antibacterial mechanism into question, indicating that further studies will be required to fully evaluate the antibacterial potential of this compound class.

Preliminary Structure-Activity Relationship Study of the MMV Pathogen Box Compound MMV675968 (2,4-Diaminoquinazoline) Unveils Novel Inhibitors of Trypanosoma brucei brucei.

New drugs are urgently needed for the treatment of human African trypanosomiasis (HAT). In line with our quest for novel inhibitors of trypanosomes, a small library of analogs of the antitrypanosomal hit (MMV675968) available at MMV as solid materials was screened for antitrypanosomal activity. In silico exploration of two potent antitrypanosomal structural analogs (7-MMV1578647 and 10-MMV1578445) as inhibitors of dihydrofolate reductase (DHFR) was achieved, together with elucidation of other antitrypanosomal modes of action. In addition, they were assessed in vitro for tentative inhibition of DHFR in a crude trypanosome extract. Their ADMET properties were also predicted using dedicated software. Overall, the two diaminoquinazoline analogs displayed approximately 40-fold and 60-fold more potency and selectivity in vitro than the parent hit, respectively (MMV1578445 (10): IC50 = 0.045 µM, SI = 1737; MMV1578467 (7): IC50 = 0.06 µM; SI = 412). Analogs 7 and 10 were also strong binders of the DHFR enzyme in silico, in all their accessible protonation states, and interacted with key DHFR ligand recognition residues Val32, Asp54, and Ile160. They also exhibited significant activity against trypanosome protein isolate. MMV1578445 (10) portrayed fast and irreversible trypanosome growth arrest between 4–72 h at IC99. Analogs 7 and 10 induced in vitro ferric iron reduction and DNA fragmentation or apoptosis induction, respectively. The two potent analogs endowed with predicted suitable physicochemical and ADMET properties are good candidates for further deciphering their potential as starting points for new drug development for HAT.

S6.5c MycetOS: identifying drugs which can penetrate the mycetoma grain

S6.5 Efforts of improving the management of mycetoma: working towards the 2030 goals, September 22, 2022, 4:45 PM - 6:15 PMMycetoma is a neglected tropical disease characterized by large subcutaneous swellings and the formation of grains. Madurella mycetomatis is the most common causative agent. Currently, mycetoma is treated with a combination of itraconazole therapy and surgery with low success rates, resulting often in amputation and social sigma. To improve the current therapeutic success rates a novel drug is needed. Due to the lack of interest in the pharmaceutical industry, an open-source drug discovery program for mycetoma was established called MycetOS. In total 1360 compounds were screened for in vitro activity against M. mycetomatis, and many more are currently being screened. Compounds that were able to inhibit growth at 100 μm, 25 μm, and had an IC50 <8 μm were selected for studying the in vivo efficacy in an M. mycetomatis grain model in the invertebrate Galleria mellonella. Out of the 1360 compounds screened against M. mycetomatis, 302 were able to inhibit growth at 100 μm, and 23 of those met all criteria to be screened in vivo. Of these 23, nine did prolong larval survival. These included 3/7 azoles tested, olorofim, fenbendazole, MMV006357, MMV022478, MMV675968, and MMV1782387. Based on these results, 6 compound series were selected for further studying, these included the fenarimols (series 1), the aminothiazoles (series 2), the phenotiazines (series 3), dihydrofolate reductase inhibitors (series 4), benzimidazoles (series 5) and the ketoximines (series 6). For series 1 in total 185 additional compounds were screened. By analyzing the in vitro activity and in vivo efficacy in relation to the chemical properties of the molecules it appears that the LogD value of a compound was important for penetrating into the mycetoma grain. In conclusion, using an open source drug discovery approach for mycetoma we were able to identify novel lead compounds. Some of these compounds were highly active against M. mycetomatis only (fenarimols, aminothiazoles, phenotiazines, and ketoximines), while other compounds such as the benzimidazoles also were active against other causative agents as well. Screening more analogs of identified compounds allowed us also to identify chemical properties which are favorable for grain penetration in vivo. This will allow us to chemically design more active compounds for this difficult to treat infection.

Anti-microbial efficacy, mechanisms and druggability evaluation of the natural flavonoids.

This study was conducted to evaluate 35 natural flavonoids for their in vitro susceptibility against E. coli (ATCC 25922), Ps. aeruginosa (ATCC 27853), B. subtilis (ATCC 530) and Staph. aureus (ATCC 6538) in search of a potential broad-spectrum antibiotic. Glabridin, a natural isoflavonoid isolated from Glycyrrhiza glabra L., was identified to be highly active with a MIC of 8-16 μg ml-1 against Staph. aureus, B. subtilis and E. coli. By the results of the docking simulation, we located the potential targets of glabridin as DNA gyrase and dihydrofolate reductase (DHFR). The subsequent DNA gyrase inhibition assays (glabridin: IC50 = 0.8516 μmol L-1 , ciprofloxacin: IC50 = 0.04697 μmol L-1 ), DHFR inhibition assays (glabridin: inhibition ratio=29%, methotrexate: inhibition ratio=45% under 100 μmol L-1 treatment) and TUNEL confirmed that glabridin acted as DNA gyrase inhibitor and DHFR mild inhibitor, exerting bactericidal activity by blocking bacterial nucleic acid synthesis. CCK-8 and in silico calculations were also conducted to verify the low cytotoxicity and acceptable druggability of glabridin. These findings suggest that glabridin represents the prototypical member of an exciting structural class of natural antimicrobial agents. This study reports a novel mechanism of bactericidal activity of glabridin against Staph. aureus.

Z. officinale-doped silver/calcium oxide nanocomposites: Catalytic activity and antimicrobial potential with molecular docking analysis

In this study, green synthesized Ag/CaO combined with a reducing agent based on a plant extract was synthesized. Then, the catalytic and antimicrobial potential of extract doped-Ag/CaO was investigated. Bio-reducing synthesis protocol was followed to fabricate Ag/CaO nanocomposites (NCs) by reducing and capping with Zingiber officinale. A series of characterization techniques was employed to examine structural, morphological and optical properties of Ag/CaO. The experimental findings favored doped nanocomposites for use as effective catalysts that degraded toxic dyes such as methylene blue and ciprofloxacin and exhibited strong antimicrobial activity against Escherichia coli (E. coli), and Staphylococcus aureus (S. aueus). Furthermore, a molecular docking investigation determined binding interaction patterns between NCs and targeted cell protein active sites. The obtained results suggested green synthesized Ag/CaO as the most effective inhibitor of dihydrofolate reductase, DNA gyrase, and FabB enzymes. Findings revealed highly economical, non-toxic, and readily available Z. officinale rhizome extract as a potential capping agent to fabricate Ag/CaO for potential applications to reduce major economic losses in the dairy industry for the first time in Pakistan.

Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV.

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions.

1-Cycloalkanecarbonyl-substituted thioureas and thiosemicarbazides as effective dihydrofolate reductase inhibitors with antibacterial activity

1-Cycloalkanecarbonyl-substituted thioureas and thiosemicarbazides as effective dihydrofolate reductase inhibitors with antibacterial activity

Microwave synthesis and antimalarial screening of novel 4-amino benzoic acid (PABA)-substituted pyrimidine derivatives as Plasmodium falciparum dihydrofolate reductase inhibitors.

The online version contains supplementary material available at 10.1007/s13205-022-03236-w.

Multiscale simulation unravels the light-regulated reversible inhibition of dihydrofolate reductase by phototrexate.

Molecular photoswitches are widely used in photopharmacology, where the biomolecular functions are photo-controlled reversibly with high spatiotemporal precision. Despite the success of this field, it remains elusive how the protein environment modulates the photochemical properties of photoswitches. Understanding this fundamental question is critical for designing more effective light-regulated drugs with mitigated side effects. In our recent work, we employed first-principles non-adiabatic dynamics simulations to probe the effects of protein on the trans to cis photoisomerization of phototrexate (PTX), a photochromic analog of the anticancer therapeutic methotrexate that inhibits the target enzyme dihydrofolate reductase (DHFR). Building upon this study, in this work, we employ multiscale simulations to unravel the full photocycle underlying the light-regulated reversible inhibition of DHFR by PTX, which remains elusive until now. First-principles non-adiabatic dynamics simulations reveal that the cis to trans photoisomerization quantum yield is hindered in the protein due to backward isomerization on the ground-state following non-adiabatic transition, which arises from the favorable binding of the cis isomer with the protein. However, free energy simulations indicate that cis to trans photoisomerization significantly decreases the binding affinity of the PTX. Thus, the cis to trans photoisomerization most likely precedes the ligand unbinding from the protein. We propose the most probable photocycle of the PTX-DHFR system. Our comprehensive simulations highlight the trade-offs among the binding affinity, photoisomerization quantum yield, and the thermal stability of the ligand's different isomeric forms. As such, our work reveals new design principles of light-regulated drugs in photopharmacology.

Structural Insight into Effective Inhibitors' Binding to Toxoplasma gondii Dihydrofolate Reductase Thymidylate Synthase.

Pyrimethamine (Pyr), a known dihydrofolate reductase (DHFR) inhibitor, has long been used to treat toxoplasmosis caused by Toxoplasma gondii (Tg) infection. However, Pyr is effective only at high doses with associated toxicity to patients, calling for safer alternative treatments. In this study, we investigated a series of Pyr analogues, previously developed as DHFR inhibitors of Plasmodium falciparum bifunctional DHFR-thymidylate synthase (PfDHFR-TS), for their activity against T. gondii DHFR-TS (TgDHFR-TS). Of these, a set of compounds with a substitution at the C6 position of the pyrimidine ring exhibited high binding affinities (in a low nanomolar range) against TgDHFR-TS and in vitro T. gondii inhibitory activity. Three-dimensional structures of TgDHFR-TS reported here include the ternary complexes with Pyr, P39, or P40. A comparison of these structures showed the minor steric strain between the p-chlorophenyl group of Pyr and Thr83 of TgDHFR-TS. Such a conflict was relieved in the complexes with the two analogues, P39 and P40, explaining their highest binding affinities described herein. Moreover, these structures suggested that the hydrophobic environment in the active-site pocket could be used for drug design to increase the potency and selectivity of antifolate inhibitors. These findings would accelerate the development of new antifolate drugs to treat toxoplasmosis.

Abstract 2334: Inhibition of DHFR modulates anti-tumor immunity

Abstract Lung cancer is the second most common type of cancer in both men and women and has the highest mortality rate. Non-small cell lung cancer (NSCLC) makes up most cases. Immune checkpoint inhibitors have significantly improved the clinical management of some NSCLC cases, yet many lung cancer patients still fail to respond to conventional immunotherapy. Therefore, more research is needed to characterize cellular pathways contribute to immune checkpoint inhibitor (ICI) resistance in NSCLC patients. Dysregulated cellular metabolism is a common feature of many cancer types, and metabolites are now known to modulate several functions inside cells such as signaling pathways and epigenetic control of gene expression. Therefore, targeting tumor metabolism may be able to combat NSCLC immunotherapy resistance. To identify potential metabolic pathways involved in the anti-tumor immune response, we employed a metabolic inhibitor-based drug screen in human NSCLC cell lines and tested for expression changes in a panel of known immune regulator genes. Notably, pharmacologic inhibition of dihydrofolate reductase (DHFR) downregulated the mRNA expression of anti-phagocytic surface protein cluster of differentiation 24 (CD24) in lung cancer cells. In line with this, shRNA knockdown and overexpression of DHFR, decreased and increased CD24 RNA and protein levels, respectively. These results show that the metabolic enzyme DHFR is involved in CD24 gene expression. Cancer cells heavily rely on DHFR for enhanced nucleotide biosynthesis to facilitate rapid tumor growth, and DHFR inhibitors are already used in the clinic for cancer therapy. This study highlights a novel interaction between reprogrammed cancer metabolism and the anti-tumor immune response, with possible clinical implications for DHFR inhibitors such as methotrexate in combination with conventional cancer immunotherapy agents. Citation Format: Austin Boese, Isabelle Young, Jihoon Kang, Courteney Malin, Jung Seok Hwang, Sumin Kang. Inhibition of DHFR modulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2334.

Multitarget, Selective Compound Design Yields Potent Inhibitors of a Kinetoplastid Pteridine Reductase 1.

The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure–activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Computational Development of Inhibitors of Plasmid-Borne Bacterial Dihydrofolate Reductase.

Resistance to trimethoprim and other antibiotics targeting dihydrofolate reductase may arise in bacteria harboring an atypical, plasmid-encoded, homotetrameric dihydrofolate reductase, called R67 DHFR. Although developing inhibitors to this enzyme may be expected to be promising drugs to fight trimethoprim-resistant strains, there is a paucity of reports describing the development of such molecules. In this manuscript, we describe the design of promising lead compounds to target R67 DHFR. Density-functional calculations were first used to identify the modifications of the pterin core that yielded derivatives likely to bind the enzyme and not susceptible to being acted upon by it. These unreactive molecules were then docked to the active site, and the stability of the docking poses of the best candidates was analyzed through triplicate molecular dynamics simulations, and compared to the binding stability of the enzyme–substrate complex. Molecule 32 ([6-(methoxymethyl)-4-oxo-3,7-dihydro-4H-pyrano[2,3-d]pyrimidin-2-yl]methyl-guanidinium) was shown by this methodology to afford extremely stable binding towards R67 DHFR and to prevent simultaneous binding to the substrate. Additional docking and molecular dynamics simulations further showed that this candidate also binds strongly to the canonical prokaryotic dihydrofolate reductase and to human DHFR, and is therefore likely to be useful to the development of chemotherapeutic agents and of dual-acting antibiotics that target the two types of bacterial dihydrofolate reductase.

Phthalide derivatives as dihydrofolate reductase inhibitors for malaria: molecular docking and molecular dynamics studies

Plasmodium falciparum dihydrofolate reductase enzyme (P. falciparum DHFR) is one of the vital drug targets for malaria treatment, as this protein is indispensable for nucleotide metabolic pathways. This research aimed to discover promising phthalide derivatives against both wild and mutant P. falciparum DHFR enzymes through various computational techniques. The binding affinities were investigated using molecular docking, which showed five compounds having the highest affinity scores against both enzymes compared to the reference compounds. MM-GBSA calculations displayed favourable free binding energy. Moreover, the ADMET properties of the compounds are within acceptable ranges. The stability of the ligand-protein complexes was studied by Molecular Dynamics (MD) simulations. Depending on the results obtained from this research, we propose three compounds to be hit against P. falciparum DHFR activity which could be examined experimentally. Communicated by Ramaswamy Sarma

Assessment of metal-based dihydrofolate reductase inhibitors on a novel mesofluidic platform

A new miniaturized micro sequential injection coupled with the lab-on-valve (µSIA–LOV) technique was full-blown to perform inhibitory studies on dihydrofolate reductase (DHFR). The system was used to evaluate the DHFR inhibition activity of metal-based anticancer compounds. The metal complexes exhibited IC50 values in the range 1.3 ± 0.3–108 ± 7 μM, with half of the complexes lying in the low μM range, i.e., 1.3 ± 0.3–4.4 ± 0.2 μM. For comparison, methotrexate (MTX), a known inhibitor of DHFR, has an IC50 value of 0.38 ± 0.06 μM. The µSIA–LOV is a versatile, robust, rapid, and easy to operate, allowing automated determination of DHFR inhibition. Moreover, the automated system requires very little sample (approximately 40 µL per analysis), uses minimal reagents (5 times less than the batch procedure used), and generates very little waste (around 1.2 mL per analysis) compared with batch methods, considerably reducing costs.