Kaurane-Type Diterpenoids as Potential Inhibitors of Dihydrofolate Reductase-Thymidylate Synthase in New World Leishmania Species.

The bifunctional enzyme Dihydrofolate reductase-thymidylate synthase (DHFR-TS) plays a crucial role in the survival of the Leishmania parasite, as folates are essential cofactors for purine and pyrimidine nucleotide biosynthesis. However, DHFR inhibitors are largely ineffective in controlling trypanosomatid infections, largely due to the presence of Pteridine reductase 1 (PTR1). Therefore, the search for structures with dual inhibitory activity against PTR1/DHFR-TS is crucial in the development of new anti-Leishmania chemotherapies. In this research, using the Leishmania major DHFR-TS recombinant protein, enzymatic inhibitory assays were performed on four kauranes and two derivatives that had been previously tested against LmPTR1. The structure 302 (6.3 µM) and its derivative 302a (4.5 µM) showed the lowest IC50 values among the evaluated molecules. To evaluate the mechanism of action of these structures, molecular docking calculations and molecular dynamics simulations were performed using a DHFR-TS hybrid model. Results showed that hydrogen bond interactions are critical for the inhibitory activity against LmDHFR-TS, as well as the presence of the p-hydroxyl group of the phenylpropanoid moiety of 302a. Finally, additional computational studies were performed on DHFR-TS structures from Leishmania species that cause cutaneous and mucocutaneous leishmaniasis in the New World (L. braziliensis, L. panamensis, and L. amazonensis) to explore the targeting potential of these kauranes in these species. It was demonstrated that structures 302 and 302a are multi-Leishmania species compounds with dual DHFR-TS/PTR1 inhibitory activity.

Identification of potential Staphylococcus aureus dihydrofolate reductase inhibitors using QSAR, molecular docking, dynamics simulations and free energy calculation.

Herein we describe the use of molecular docking simulations, quantitative structure-activity relationships studies and ADMETox predictions to analyse the molecular recognition of a series of 7-aryl-2,4-diaminoquinazoline derivatives on the inhibition of Staphylococcus aureus dihydrofolate reductase and conducted a virtual screening to discover new potential inhibitors. A quantitative structure-activity relationship model was developed using 40 compounds and two selected descriptors. These descriptors indicated the importance of pKa and molar refractivity for the inhibitory activity against SaDHFR. The values of R2train, CVLOO and R2test generated by the model were 0.808, 0.766, and 0.785, respectively. The integration between QSAR, molecular docking, ADMETox analysis and molecular dynamics simulations with binding free energies calculation, yielded the compounds PC-124127620, PC-124127795 and PC-124127805 as promising candidates to SaDHFR inhibitors. These compounds presented high potency, good pharmacokinetics and toxicological profile. Thus, these molecules are good potential antimicrobial agent to treatment of infect disease caused by S. aureus.Communicated by Ramaswamy H. Sarma.

Machine-Learning- and Structure-Based Virtual Screening for Selecting Cinnamic Acid Derivatives as Leishmania major DHFR-TS Inhibitors.

The critical enzyme dihydrofolate reductase-thymidylate synthase in Leishmania major (LmDHFR-TS) serves a dual-purpose role and is essential for DNA synthesis, a cornerstone of the parasite's reproductive processes. Consequently, the development of inhibitors against LmDHFR-TS is crucial for the creation of novel anti-Leishmania chemotherapies. In this study, we employed an in-house database containing 314 secondary metabolites derived from cinnamic acid that occurred in the Asteraceae family. We conducted a combined ligand/structure-based virtual screening to identify potential inhibitors against LmDHFR-TS. Through consensus analysis of both approaches, we identified three compounds, i.e., lithospermic acid (237), diarctigenin (306), and isolappaol A (308), that exhibited a high probability of being inhibitors according to both approaches and were consequently classified as promising hits. Subsequently, we expanded the binding mode examination of these compounds within the active site of the test enzyme through molecular dynamics simulations, revealing a high degree of structural stability and minimal fluctuations in its tertiary structure. The in silico predictions were then validated through in vitro assays to examine the inhibitory capacity of the top-ranked naturally occurring compounds against LmDHFR-TS recombinant protein. The test compounds effectively inhibited the enzyme with IC50 values ranging from 6.1 to 10.1 µM. In contrast, other common cinnamic acid derivatives (i.e., flavonoid glycosides) from the Asteraceae family, such as hesperidin, isovitexin 4'-O-glucoside, and rutin, exhibited low activity against this target. The selective index (SI) for all tested compounds was determined using HsDHFR with moderate inhibitory effect. Among these hits, lignans 306 and 308 demonstrated the highest selectivity, displaying superior SI values compared to methotrexate, the reference inhibitor of DHFR-TS. Therefore, continued research into the anti-leishmanial potential of these C6C3-hybrid butyrolactone lignans may offer a brighter outlook for combating this neglected tropical disease.

Identification of Active Compounds against Melanoma Growth by Virtual Screening for Non-Classical Human DHFR Inhibitors.

Antifolates such as methotrexate (MTX) have been largely known as anticancer agents because of their role in blocking nucleic acid synthesis and cell proliferation. Their mechanism of action lies in their ability to inhibit enzymes involved in the folic acid cycle, especially human dihydrofolate reductase (hDHFR). However, most of them have a classical structure that has proven ineffective against melanoma, and, therefore, inhibitors with a non-classical lipophilic structure are increasingly becoming an attractive alternative to circumvent this clinical resistance. In this study, we conducted a protocol combining virtual screening (VS) and cell-based assays to identify new potential non-classical hDHFR inhibitors. Among 173 hit compounds identified (average logP = 3.68; average MW = 378.34 Da), two-herein, called C1 and C2-exhibited activity against melanoma cell lines B16 and A375 by MTT and Trypan-Blue assays. C1 showed cell growth arrest (39% and 56%) and C2 showed potent cytotoxic activity (77% and 51%) in a dose-dependent manner. The effects of C2 on A375 cell viability were greater than MTX (98% vs 60%) at equivalent concentrations and times. Our results indicate that the integrated in silico/in vitro approach provided a benchmark to identify novel promising non-classical DHFR inhibitors showing activity against melanoma cells.

A Target Engagement Assay to Assess Uptake, Potency, and Retention of Antibiotics in Living Bacteria.

New antibiotics are urgently needed to counter the emergence of antimicrobial-resistant pathogenic bacteria. A major challenge in antibiotic drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetic binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity. We validated this approach using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of target engagement assays in bacteria to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.

Dihydrofolate Reductase (DHFR) del19bp Polymorphism and Down Syndrome Offspring.

Down syndrome (DS) is the most common form of mental disability of genetic etiology. Nondisjunction of chromosome 21 is the leading cause of the syndrome. In general, free trisomy 21 cases originate from missegregation in maternal meiosis. Several reports have suggested an association between genetic variants in genes encoding folate metabolizing enzymes and the predisposition to chromosome missegregation. We have conducted a case-control study of 109 DS case mothers (MDS) and 248 control mothers (CM) to assess the association between DHFR del19bp polymorphism and an increased risk of bearing a DS child. Genomic DNA was extracted from buccal cells, and molecular analysis of DHFR del19pb polymorphism was performed by polymerase chain reaction (PCR). Both MDS and CM allelic and genotypic distributions were in Hardy-Weinberg equilibrium. The frequency of DHFR del19pb-mutated allele was 0.54 in MDS and 0.46 in CM. Overall analysis showed that the mutant allele was borderline associated with DS risk (OR 1.38; 95% CI 1.00-1.89; P = 0.05) and a weak positive association for del/del and/or wt/del genotypes of DHFR del19pb polymorphism compared to homozygous wt/wt genotype was identified (OR = 1.75; 95% CI 1.01-3.03; P = 0.05). When we have analyzed data stratified by age, there is an increased risk of bearing a DS child associated with the polymorphic allele (OR = 1.49; 95% CI 1.03-2.16; P = 0.03), suggesting that DHFR del 19-bp polymorphism could be an independent risk factor for DS in women aged < 40 years old.

Identification, Characterization and Molecular Modelling Studies of Schistosoma mansoni Dihydrofolate Reductase Inhibitors: From Assay Development to Hit Identification.

INTRODUCTION: Schistosoma mansoni is responsible for virtually all reported cases of schistosomiasis in Latin America and the emergence of praziquantel- and oxaminiquine-resistant strains makes it urgent to develop new schistosomicide agents. Dihydrofolate reductases (DHFR) from bacteria and protozoan parasites are considered validated macromolecular targets for this goal, but S. mansoni DHFR (SmDHFR) has been largely overlooked. To fill this gap in knowledge, the present work describes optimized conditions to carry out thermal shift assays with SmDHFR, as well as a balanced kinetic assay that supports 2,4-diaminopyrimidine derivatives as SmDHFR inhibitors. The most potent inhibitor (2a) shows a large shift of the melting temperature (ΔTm = + 8 ± 0,21 ºC) and a low micromolar IC50 value (12 ± 2,3 µM). Both thermal shift and classical kinectic assay suggest that 2a binds to the substrate binding site (competitive inhibition mechanism). This information guided docking and molecular dynamics studies that probed 2a interaction profile towards SmDHFR. CONCLUSION: In conclusion, this work not only provides standardized assay conditions to identify SmDHFR inhibitors, but also describes the binding profile of the first low micromolar inhibitor of this macromolecular target.

Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase.

The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.

Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia.

BACKGROUND: Non-synonymous mutations in dhfr and dhps genes in Plasmodium vivax are associated with sulfadoxine-pyrimethamine (SP) resistance. The present study aimed to assess the prevalence of point mutations in P. vivax dhfr (pvdhfr) and P. vivax dhps (pvdhps) genes in three countries: Lao PDR, India and Colombia. METHODS: Samples from 203 microscopically diagnosed vivax malaria were collected from the three countries. Five codons at positions 13, 57, 58, 61, and 117 of pvdhfr and two codons at positions 383 and 553 of pvdhps were examined by polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS: The largest number of 58R/117 N double mutations in pvdhfr was observed in Colombia (94.3 %), while the corresponding wild-type amino acids were found at high frequencies in Lao PDR during 2001-2004 (57.8 %). Size polymorphism analysis of the tandem repeats within pvdhfr revealed that 74.3 % of all the isolates carried the type B variant. Eighty-nine per cent of all the isolates examined carried wild-type pvdhps A383 and A553. CONCLUSIONS: Although SP is not generally used to treat P. vivax infections, mutations in dhfr and dhps that confer antifolate resistance in P. vivax are common. The data strongly suggest that, when used primarily to treat falciparum malaria, SP can exert a substantial selective pressure on P. vivax populations, and this can lead to point mutations in dhfr and dhps. Accurate data on the global geographic distribution of dhfr and dhps genotypes should help to inform anti-malarial drug-use policies.

The 19-bp deletion polymorphism of dihydrofolate reductase (DHFR) and nonsyndromic cleft lip with or without cleft palate: evidence for a protective role

Objective Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) are among the most common congenital birth defects worldwide. Several lines of evidence point to the involvement of folate, as well as folate metabolizing enzymes in risk reduction of orofacial clefts. Dihydrofolate reductase (DHFR) enzyme participates in the metabolic cycle of folate and has a crucial role in DNA synthesis, a fundamental feature of gestation and development. A functional polymorphic 19-bp deletion within intron-1 of DHFR has been associated with the risk of common congenital malformations. The present study aimed to evaluate the possible association between DHFR 19-bp deletion polymorphism and susceptibility to NS-CL/P in an Iranian population. Material and Methods The current study recruited 100 NS-CL/P patients and 100 healthy controls. DHFR 19-bp deletion was determined using an allele specific-PCR method. Results We observed the DHFR 19-bp homozygous deletion genotype (D/D) vs. homozygous wild genotype (WW) was more frequent in controls than in NS-CL/P patients (25% vs. 13%), being associated with a reduced risk of NS-CL/P in both codominant (OR=0.33, P=0.027) and recessive (OR=0.45, P=0.046) tested inheritance models. We also stratified the cleft patients and reanalyzed the data. The association trend for CL+CL/P group compared to the controls revealed that the DD genotype in both codominant (OR=0.30, P=0.032) and recessive models (OR=0.35, P=0.031) was associated with a reduced risk of CL+CL/P. Conclusions Our results for the first time suggested the DHFR 19-bp D/D genotype may confer a reduced risk of NS-CL/P and might act as a protective factor against NS-CL/P in the Iranian subjects. .

A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (ß-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.

Haplotypes associated with resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum in two malaria endemic locations in Colombia.

Colombia has four main malaria transmission zones. In vivo efficacy studies carried out in these areas showed big differences in the response of Plasmodium falciparum to treatment with sulphadoxine-pyrimethamine. In addition, there is still insufficient information about the genetics of P. falciparum populations. The objective of this study was to determine the haplotypes in dhfr and dhps genes of P. falciparum circulating in two distinct endemic zones. Samples from patients with non-complicated P. falciparum malaria were collected: 135 from Tumaco and 206 from Tierralta. Alleles 108 and 51 of the dhfr gene, and 437 and 540 of the dhps gene were analyzed by PCR/enzymatic restriction, while alleles 59 and 164 (dhfr), and 581(dhps) by PCR/dot blot/hybridization. Five different haplotypes were found, of which the triple mutant 51I/C59/108N/I164/437G/K540/A581 was the most frequent (54.6%). In Tumaco, the parasites with wild haplotype predominated, while mutant parasites predominated in Tierralta. Another interesting finding is the presence of the C59R mutation in the dhfr gene in two samples, a mutation rarely found in South America. These data provide information about parasite population genetics and highlight the importance of starting a long term molecular surveillance program.

Status of dhps and dhfr genes of Plasmodium falciparum in Colombia before artemisinin based treatment policy / Estado de los genes dhps y dhfr de Plasmodium falciparum en Colombia antes de la recomendación de tratamiento basado en artemisinina

Introduction: Surveillance of the genetic characteristics of dhps and dhfr can be useful to outline guidelines for application of intermittent preventive therapy in Northwest Colombia and to define the future use of antifolates in artemisinin-based combination therapy schemes. Objective: To evaluate the frequency of mutations in dhps and dhfr and to characterize parasite populations using msp-1, msp-2 and glurp in historic samples before artemisinin-based therapy was implemented in the country. Methods: A controlled clinical study was carried out on randomly selected Plasmodium falciparum infected volunteers of Northwest Colombia (Turbo and Zaragoza). A sample size of 25 subjects per region was calculated. Treatment efficacy to antifolates was assessed. Molecular analyses included P. falciparum genotypes by msp-1, msp-2 and glurp and evaluation of the status of codons 16, 51, 59, 108 and 164 of dhfr and 436, 437, 540, 581 and 613 of dhps. Results: In total 78 subjects were recruited. A maximum number of 4 genotypes were detected by msp-1, msp-2 and glurp. Codons 16, 59 and 164 of the dhfr gene exhibited the wild-type form, while codons 51 and 108 were mutant. In the dhps gene, the mutant 437 glycine was detected in 85% on day 0, while codons 436, 540, 581 and 613 were wild-type. Conclusions: Plasmodium falciparum populations were very homogeneous in this region of Colombia, and the triple mutants of dhfr and dhps Asn108, Ile51 and Gly437 were predominant in clinical isolates.

Introducción. La vigilancia de las características genéticas de dhps y dhfr puede utilizarse para delinear guías de aplicación de terapia preventiva intermitente en el nordeste de Colombia y para definir el uso futuro de los antifolatos en esquemas terapéuticos basados en artemisinina. Objetivo. Evaluar la frecuencia de mutaciones en dhps y dhfr, y caracterizar las poblaciones parasitarias usando msp-1, msp-2 y glurp, en muestras históricas obtenidas antes de la implementación en el país de la terapia basada en artemisinina. Métodos. Se llevó a cabo un estudio clínico controlado en voluntarios infectados con Plasmodium falciparum seleccionados aleatoriamente y provenientes del nordeste de Colombia (Turbo y Zaragoza). Se calculó una muestra de 25 sujetos por región. Se evaluó la eficacia al tratamiento con antifolatos. Los análisis moleculares incluyeron la obtención de genotipos de msp-1, msp-2 y glurp y el estado de los codones 16, 51, 59, 108 y 164 de dhfr, y 436, 437, 5540, 581 y 613 de dhps. Resultados. Se estudiaron 78 sujetos. Se detectó un número máximo de 4 genotipos con msp-1, msp-2 y glurp. Los codones 16, 59 y 164 del gen dhfr se encontraron en su forma silvestre, mientras que los codones 51 y 108 estaban mutados. En el gen dhps, la forma mutante (glicina) en el codón 437, se detectó en 85% el día 0, mientras que los codones 436, 540, 581 y 613 se encontraron silvestres. Conclusiones. Las poblaciones de P. falciparum son muy homogéneas en esta región de Colombia y las triple mutantes de dhfr y dhps Asn108, Ile51 and Gly437, predominaron en los aislamientos clínicos.