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Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid 'P' within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest kcat of 0.95 s−1, followed by rDHFR with 0.14 s−1, while eDHFR displayed the lowest kcat of 0.09 s−1. Remarkably, the K65P variant induced a significant reduction in Km, resulting in a 1.8-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue. [ABSTRACT FROM AUTHOR]

Lung cancer is a leading cause of cancer-related death worldwide that needs updated therapies to contrast both the serious side effects and the occurrence of drug resistance. A panel of non-small cell lung cancer (NSCLC) cells were herein employed as cancer models. Eight structurally related gold(I) and gold(III) complexes with NHC and halides or triphenylphosphane ligands were investigated as lung cancer cell growth inhibitors. As expected, gold compounds with PPh3 were found to be more cytotoxic than homoleptic [(NHC)2-Au(I)]X or heteroleptic NHC-Au(I)X or NHC-Au(III)X3 complexes. Mixed ligand gold(I) compounds exhibiting the linear NHC-AuPPh3 (compound 7) or the trigonal NHC-Au(Cl)PPh3 (compound 8) arrangements at the central metal were found to be the best lung cancer cytotoxic compounds. Analysis of the TrxR residual activity of the treated cells revealed that these compounds efficiently inhibit the most accredited molecular target for gold compounds, the TrxR, with compound 8 reaching more than 80% activity reduction in lung cells. Some of the current cancer lung therapy protocols consist of specific lung cancer cell cytotoxic agents combined with antifolate drugs; interestingly, the herein gold compounds are both TrxR and antifolate inhibitors. The human DHFR was inhibited with IC50 ranging between 10–21 µM, depending on substrate concentrations, proceeding by a likely allosteric mechanism only for compound 8. [ABSTRACT FROM AUTHOR]

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism. [ABSTRACT FROM AUTHOR]

Malaria remains a global health challenge with significant mortality and morbidity annually, with resistant parasite strains complicating treatment efforts. There is an acute need for novel antimalarial drugs that can put a stop to the future public health crisis caused by the multi-drug resistance strains of the Plasmodium parasite. However, the discovery of these new components is very challenging in the context of the generation of multi-drug resistance properties of malaria. The novel drugs also need to have several properties involving enhanced therapeutic prospects, successful treatment capabilities, and novel mechanisms of action that will forestall the resistance. To successfully achieve this aim researchers are trying to focus on exploring promising malaria targets. Various approaches have been made for the development of drugs for malaria including the remodelling of existing drugs and the development of novel inhibitors which acts on new targets. Advancement in the study provides more information on the biology of parasites and the new targets which help in the development of novel drugs. The present review focuses on the study of novel targets of malaria parasites and subsequent inhibitors of those particular targets. Some of these targets include malarial protease, various transporter proteins, enzymes involved in the synthesis of DNA, and nucleic acids like dihydroorotate dehydrogenase, dihydrofolate reductase, apicoplast and dihydropteroate synthase. Other potential targets are also included in this review such as isoprenoid biosynthesis, farnesyl transferase of parasite, P. falciparum translational elongation factor 2, and phosphatidyl inositol 4 kinase. These promising targets have also been summed up along with their corresponding inhibitors for combating multi-drug resistance malaria. [ABSTRACT FROM AUTHOR]

Viral communities exist in a variety of ecosystems and play significant roles in mediating biogeochemical processes, whereas viruses inhabiting strongly alkaline geochemical systems remain underexplored. In this study, the viral diversity, potential functionalities, and virus-host interactions in a strongly alkaline environment (pH = 10.4-12.4) exposed to the leachates derived from the serpentinization-like reactions of smelting slags were investigated. The viral populations (e.g., Herelleviridae, Queuovirinae, and Inoviridae) were closely associated with the dominating prokaryotic hosts (e.g., Meiothermus, Trueperaceae, and Serpentinomonas) in this ultrabasic environment. Auxiliary metabolic genes (AMGs) suggested that viruses may enhance hosts' fitness by facilitating cofactor biosynthesis, hydrogen metabolism, and carbon cycling. To evaluate the activity of synthesis of essential cofactor vitamin B9 by the viruses, a viral folA (vfolA) gene encoding dihydrofolate reductase (DHFR) was introduced into a thymidineauxotrophic strain Escherichia coli MG1655 ΔfolA mutant, which restored the growth of the latter in the absence of thymidine. Notably, the homologs of the validated vDHFR were globally distributed in the viromes across various ecosystems. The present study sheds new light on the unique viral communities in hyperalkaline ecosystems and their potential beneficial impacts on the coexisting microbial consortia by supplying essential cofactors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Introduction: Cancer is the second most prevalent cause of mortality in the world, despite the availability of several medications for cancer treatment. Therefore, the cancer research community emphasized on computational techniques to speed up the discovery of novel anticancer drugs. Methods: In the current study, QSAR-based virtual screening was performed on the Zinc15 compound library (271 derivatives of methotrexate (MTX) and phototrexate (PTX)) to predict their inhibitory activity against dihydrofolate reductase (DHFR), a potential anticancer drug target. The deep learning-based ADMET parameters were employed to generate a 2D QSAR model using the multiple linear regression (MPL) methods with Leave-one-out cross-validated (LOO-CV) Q2 and correlation coefficient R2 values as high as 0.77 and 0.81, respectively. Results: From the QSAR model and virtual screening analysis, the top hits (09,27, 41,68,74,85,99,180) exhibited pIC50 ranging from 5.85 to 7.20 with a minimum binding score of -11.6 to -11.0 kcal/mol and were subjected to further investigation. The ADMET attributes using the message-passing neural network (MPNN) model demonstrated the potential of selected hits as an oral medication based on lipophilic profile Log P (0.19-2.69) and bioavailability (76.30% to 78.46%). The clinical toxicity score was 31.24% to 35.30%, with the least toxicity score (8.30%) observed with compound 180. The DFT calculations were carried out to determine the stability, physicochemical parameters and chemical reactivity of selected compounds. The docking results were further validated by 100 ns molecular dynamic simulation analysis. Conclusion: The promising lead compounds found endorsed compared to standard reference drugs MTX and PTX that are best for anticancer activity and can lead to novel therapies after experimental validations. Furthermore, it is suggested to unveil the inhibitory potential of identified hits via in-vitro and in-vivo approaches. [ABSTRACT FROM AUTHOR]

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism.

Introduction: Cancer is the second most prevalent cause of mortality in the world, despite the availability of several medications for cancer treatment. Therefore, the cancer research community emphasized on computational techniques to speed up the discovery of novel anticancer drugs.Methods: In the current study, QSAR-based virtual screening was performed on the Zinc15 compound library (271 derivatives of methotrexate (MTX) and phototrexate (PTX)) to predict their inhibitory activity against dihydrofolate reductase (DHFR), a potential anticancer drug target. The deep learning-based ADMET parameters were employed to generate a 2D QSAR model using the multiple linear regression (MPL) methods with Leave-one-out cross-validated (LOO-CV) Q2 and correlation coefficient R2 values as high as 0.77 and 0.81, respectively.Results: From the QSAR model and virtual screening analysis, the top hits (09, 27, 41, 68, 74, 85, 99, 180) exhibited pIC50 ranging from 5.85 to 7.20 with a minimum binding score of -11.6 to -11.0 kcal/mol and were subjected to further investigation. The ADMET attributes using the message-passing neural network (MPNN) model demonstrated the potential of selected hits as an oral medication based on lipophilic profile Log P (0.19-2.69) and bioavailability (76.30% to 78.46%). The clinical toxicity score was 31.24% to 35.30%, with the least toxicity score (8.30%) observed with compound 180. The DFT calculations were carried out to determine the stability, physicochemical parameters and chemical reactivity of selected compounds. The docking results were further validated by 100 ns molecular dynamic simulation analysis.Conclusion: The promising lead compounds found endorsed compared to standard reference drugs MTX and PTX that are best for anticancer activity and can lead to novel therapies after experimental validations. Furthermore, it is suggested to unveil the inhibitory potential of identified hits via in-vitro and in-vivo approaches.

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.

Recent work from Nguyen et al. unveils massively parallel measurements of epistatic interactions between two enzymes, dihydrofolate reductase and thymidylate synthase, in their natural cellular context. Almost 3000 mutations of DHFR in three TYMS backgrounds reveal a complex interaction network. The authors capture much of this complexity using a simple model. [ABSTRACT FROM AUTHOR]

In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1C1039G/+ Marfan Syndrome (MFS) mice. The expression of mature/active TGFβ and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1C1039G/+ mice. In vivo treatment with anti-TGFβ completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1C1039G/+ mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H4B. Notably, circulating H4B levels were accurately predictive of tissue H4B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H4B for TAA. Furthermore, FA diet abrogated TGFβ and NOX4 expression, disrupting the feed-forward loop to inactivate TGFβ/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H4B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1C1039G/+ mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFβ, indicating TGFβ-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFβ and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.

Nutritive symbiosis between bacteria and ticks is observed across a range of ecological contexts; however, little characterization on the molecular components responsible for this symbiosis has been done. Previous studies in our lab demonstrated that Rickettsia monacensis str. Humboldt (strain Humboldt) can synthesize folate de novo via the folate biosynthesis pathway involving folA, folC, folE, folKP, and ptpS genes. In this study, expression of the strain Humboldt folA gene within a folA mutant Escherichia coli construct was used to functionally characterize the strain Humboldt folA folate gene in vivo. The strain Humboldt folA folate gene was subcloned into a TransBac vector and transformed into a folA mutant E. coli construct. The mutant containing strain Humboldt folA subclone and a pFE604 clone of the knocked-out folA gene was cured of pFE604. Curing of the folA mutant E. coli construct was successful using acridine orange and 43.5 °C incubation temperature. The plasmid curing assay showed curing efficiency of the folA mutant at 100%. Functional complementation was assessed by growth phenotype on minimal media with and without IPTG between strain Humboldt folA and E. coli folA. Large and homogenous wild-type colony growth was observed for both strain Humboldt and E. coli folA on minimal media with 0.1 mM IPTG, wild-type growth for strain Humboldt folA and pin-point growth for E. coli folA on 0.01 mM IPTG, and pin-point growth without IPTG for both strain Humboldt and E. coli folA. This study provides evidence substantiating the in vivo functionality of strain Humboldt folA in producing functional gene products for folate biosynthesis.

Candida auris has emerged as a global health problem with a dramatic spread by nosocomial transmission and a high mortality rate. Antifungal therapy for C. auris infections is currently limited due to widespread resistance to fluconazole and amphotericin B and increasing resistance to the front‐line drug echinocandin. Therefore, new treatments are urgently required to combat this pathogen. Dihydrofolate reductase (DHFR) has been validated as a potential drug target for Candida species, although no structure of the C. auris enzyme (CauDHFR) has been reported. Here, crystal structures of CauDHFR are reported as an apoenzyme, as a holoenzyme and in two ternary complexes with pyrimethamine and cycloguanil, which are common antifolates, at near‐atomic resolution. Preliminary biochemical and biophysical assays and antifungal susceptibility testing with a variety of classical antifolates were also performed, highlighting the enzyme‐inhibition rates and the inhibition of yeast growth. These structural and functional data might provide the basis for a novel drug‐discovery campaign against this global threat. [ABSTRACT FROM AUTHOR]

Human dihydrofolate reductase (hDHFR) inhibitors have been a popular research object designed as anti‐cancer, anti‐malarial, and antibacterial drugs for decades. Besides quantitative structure‐activity relationship (QSAR), artificial intelligence (AI) has recently been introduced in numerous professional biological researches, such as molecular drug design and biological activity prediction. In this study, we construct a deep‐learning workflow for designing novel hDHFR inhibitors. This workflow mainly includes two networks, as described in the following: The first one is the artificial neural network trained by the molecules selected from the ChEMBL database with experimental hDHFR inhibitions as the label to evaluate the bioactivity of the designed molecular structures constructed from the second network. The second network utilizes conditional generative and adversarial networks (cGAN) to generate candidate molecules with the desired properties. Finally, the obtained candidate molecules with high hDHFR inhibition are subjected to a molecular docking process to verify their binding patterns and affinity strengths inside the active site of hDHFR. In the end, we have successfully identified several novel drug‐like compounds with hDHFR inhibition comparable to those currently used in clinics. We present a new tool to effectively design new drug‐like compounds through an AI approach. [ABSTRACT FROM AUTHOR]

Sulfadoxine-pyrimethamine (SP) is no longer recommended for the treatment of uncomplicated falciparum malaria due to its increasing failure rate, but is still being used as preventive treatment during pregnancy and in chemoprevention. SP targets either dihydrofolate reductase (DHFR) and dihydroptereoate synthase (DHPS) involved in folate biosynthesis in the parasite. This study screened for mutation in SP target genes, dhfr and dhps in Plasmodium falciparum among out-patients in Awka South, Anambra State. Blood samples were obtained from 210 patients aged 5-64 years with febrile conditions. The blood samples were screened for malaria parasites using blood smear stained with giemsa. The dhfr and dhps genes were amplified using PCR and sequenced using dideoxy chain termination method. The overall prevalence of Plasmodium falciparum malaria in this study was 74.8 % (157/210) out of which 19.1 % (30/157) had high parasite count of = 4000 asexual parasite/µL of blood. Out of this number, 14.6% [23/157] with high yield and purity of =50 ng/µL and =1.7 respectively were considered good for downstream PCR. The result showed that dhfr alone was detected in 8/23 [34%], dhps alone was seen in 7/23 [30%] while both dhfr and dhps genes were seen in 4/30 [13%]. In all [8/23] parasites where dhfr was detected, mutations at Asn-108, Ile-51 and Arg-59 were present. Additional mutation was seen in 75% [6/8] at position Leu-164. Gly-581, Phe-436 and Thr-613 mutations were seen in all isolates where dhps were detected. Rare Leu-164 and Thr-613 mutants were seen in dhfr and dhps respectively. These mutants may be responsible for SP resistance in this region, suggesting an increased compromise over the efficacy of SP in malaria treatment. [ABSTRACT FROM AUTHOR]

Introduction: Endothelial nitric oxide synthase (eNOS) uncoupling plays a significant role in acute vasoconstriction during early brain injury (EBI) after subarachnoid hemorrhage (SAH). Astrocytes in the neurovascular unit extend their foot processes around endothelia. In our study, we tested the hypothesis that increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression in astrocytes after SAH leads to eNOS uncoupling. Methods:We utilized laser speckle contrast imaging for monitoring cortical blood flow changes in mice, nitric oxide (NO) kits to measure the level of NO, and a coculture system to study the effect of astrocytes on endothelial cells. Moreover, the protein levels were assessed by Western blot and immunofluorescence staining. We used CCK-8 to measure the viability of astrocytes and endothelial cells, and we used the H2O2 kit to measure the H2O2 released from astrocytes. We used GSK2795039 as an inhibitor of NOX2, whereas lentivirus and adeno-associated virus were used for dihydrofolate reductase (DHFR) knockdown in vivo and in vitro. Results: The expression of NOX2 and the release of H2O2 in astrocytes are increased, which was accompanied by a decrease in endothelial DHFR 12 h after SAH. Moreover, the eNOS monomer/dimer ratio increased, leading to a decrease in NO and acute cerebral ischemia. All of the above were significantly alleviated after the administration of GSK2795039. However, after knocking down DHFR both in vivo and in vitro, the protective effect of GSK2795039 was greatly reversed. Discussion: The increased level of NOX2 in astrocytes contributes to decreased DHFR in endothelial cells, thus aggravating eNOS uncoupling, which is an essential mechanism underlying acute vasoconstriction after SAH. [ABSTRACT FROM AUTHOR]

A series of Michael adducts of malononitrile and sulfonamide chalcones were synthesized, characterized, and evaluated for their antifilarial activity. Out of 14 compounds, N-(4-(4,4-dicyano-3-p-tolylbutanoyl)phenyl)benzenesulfonamide showed favorable drug-likeness properties with marked antifilarial effects at micro-molar dosages. Apoptosis in Brugia malayi microfilariae was confirmed by EB/AO staining, MTT assay, and cytoplasmic cytochrome c ELISA. Since chalcone and folate synthesis pathways share the same substrate, we hypothesize a structural analogy-based inhibition of folate metabolism by this compound. Molecular docking against a pre-validated BmDHFR protein showed more favorable thermodynamic parameters than a positive control, epicatechin-3-gallate. The compound significantly suppressed the DHFR activity in a parasite extract in vitro. Our hypothesis is also supported by a significant reversal of DHFR inhibition by folate addition, which indicated a plausible mechanism of competitive inhibition. These results demonstrate that targeting filarial folate metabolism through DHFR with consequent apoptosis induction might be rewarding for therapeutic intervention. This study reveals a novel rationale of the structural analogy-based competitive inhibition of DHFR by Michael adducts of sulfonamide chalcones. [ABSTRACT FROM AUTHOR]

Cellulitis is a skin disease which is caused by the pathogen Staphylococcus aureus. The protein Dihydrofolate reductase is an important drug based target for Trimethoprim. Dihydrofolate reductase is reversibly inhibited by the drug trimethoprim. One of the major enzymes responsible for converting dihydrofolic acid into tetrahydrofolic acid. The screening of microbial natural products, Soil isolation is a rich source of actinomycetes. The actinomycetes sp. KK21-2 strain isolated from the soil, the aqueous extract of the isolates yielded 33 active metabolites from the LC-MS analysis. Based on the ADMETox analysis, 8 compounds were excluded and finally 25 compounds were selected for the docking analysis. Protein structure of S. aureus retrieved from PDB, drug and compounds were obtained from the PubChem Database. Molecular docking (XP) and Induced Fit Docking (IFD) were completed utilizing the Glide Module of Schrodinger. Molecular dynamics simulation employing the Schrodinger Desmond module was used to examine the stability of the docked complex. From the results of docking analysis and MD Simulation result 5-(3,4-Dihydroxy-1,5-cyclohexadien-1-yl) phenylhydantoin have suggest this compound have multi- targeting potency against the cellulitis skin disease from the pathogen Staphylococcus aureus, in future need to be verified through in vitro and in vivo analysis.

Enzyme function and evolution are influenced by the larger context of a metabolic pathway. Deleterious mutations or perturbations in one enzyme can often be compensated by mutations to others. We used comparative genomics and experiments to examine evolutionary interactions with the essential metabolic enzyme dihydrofolate reductase (DHFR). Analyses of synteny and co-occurrence across bacterial species indicate that DHFR is coupled to thymidylate synthase (TYMS) but relatively independent from the rest of folate metabolism. Using quantitative growth rate measurements and forward evolution in Escherichia coli, we demonstrate that the two enzymes adapt as a relatively independent unit in response to antibiotic stress. Metabolomic profiling revealed that TYMS activity must not exceed DHFR activity to prevent the depletion of reduced folates and the accumulation of the intermediate dihydrofolate. Comparative genomics analyses identified >200 gene pairs with similar statistical signatures of modular co-evolution, suggesting that cellular pathways may be decomposable into small adaptive units.

Hypertension and abdominal aortic aneurysm (AAA) are severe cardiovascular diseases with incompletely defined molecular mechanisms. In the current study we generated dihydrofolate reductase (DHFR) knockout mice for the first time to examine its potential contribution to the development of hypertension and AAA, as well as the underlying molecular mechanisms. Whereas the homozygote knockout mice were embryonically lethal, the heterozygote knockout mice had global reduction in DHFR protein expression and activity. Angiotensin II infusion into these animals resulted in substantially exaggerated elevation in blood pressure and development of AAA, which was accompanied by excessive eNOS uncoupling activity (featured by significantly impaired tetrahydrobiopterin and nitric oxide bioavailability), vascular remodeling (MMP2 activation, medial elastin breakdown and adventitial fibrosis) and inflammation (macrophage infiltration). Importantly, scavenging of mitochondrial reactive oxygen species with Mito-Tempo in vivo completely abrogated development of hypertension and AAA in DHFR knockout mice, indicating a novel role of mitochondria in mediating hypertension and AAA downstream of DHFR deficiency-dependent eNOS uncoupling. These data for the first time demonstrate that targeting DHFR-deficiency driven mitochondrial dysfunction may represent an innovative therapeutic option for the treatment of AAA and hypertension.

A polymer-based electrode capable of specific detection of human serum albumin, and its glycated derivatives, is described. The sensor is constructed from a glass microscope slide coated with a synthesized, polythiophene film bearing a protected, iminodiacetic acid motif. The electrode surface is then further elaborated to a functional biosensor through deprotection of the iminodiacetic acid, followed by metal-affinity immobilization of a specific and high-affinity, albumin ligand. Albumin was then quantified in buffer and synthetic urine via electrochemical impedance spectroscopy. Glycated albumin was next bound to a boronic acid-modified, single-cysteine dihydrofolate reductase variant to quantify glycation ratios by square-wave voltammetry. The platform offers high sensitivity, specificity, and reproducibility in an inexpensive arrangement. The detection limits exceed the requirements for intermediate-term glycemic control monitoring in diabetes patients at 5 and 1 nM for albumin and its glycated forms, respectively.

We have shown that hydrogen peroxide (H2O2) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H2O2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H2O2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H2O2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H2O2 on DHFR promoter activity. H2O2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H2O2. E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)-infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H2O2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.

Dihydrofolate reductase (DHFR) is a crucial enzyme that maintains the levels of 5,6,7,8-tetrahydrofolate (THF) required for the biological synthesis of the building blocks of DNA, RNA, and proteins. Over-activation of DHFR results in the progression of multiple pathological conditions such as cancer, bacterial infection, and inflammation. Therefore, DHFR inhibition plays a major role in treating these illnesses. Sesquiterpenes of various types are prime metabolites derived from the marine sponge Dactylospongia elegans and have demonstrated antitumor, anti-inflammation, and antibacterial capacities. Here, we investigated the in silico potential inhibitory effects of 87 D. elegans metabolites on DHFR and predicted their ADMET properties. Compounds were prepared computationally for molecular docking into the selected crystal structure of DHFR (PDB: 1KMV). The docking scores of metabolites 34, 28, and 44 were the highest among this series (gscore values of −12.431, −11.502, and −10.62 kcal/mol, respectively), even above the co-crystallized inhibitor SRI-9662 score (−10.432 kcal/mol). The binding affinity and protein stability of these top three scored compounds were further estimated using molecular dynamic simulation. Compounds 34, 28, and 44 revealed high binding affinity to the enzyme and could be possible leads for DHFR inhibitors; however, further in vitro and in vivo investigations are required to validate their potential. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation. [ABSTRACT FROM AUTHOR]

Tetrahydrobiopterin (BH4) is a vital coenzyme for several enzymes involved in diverse enzymatic reactions in animals. BH4 deficiency can lead to metabolic and neurological disorders due to dysfunction in its metabolism. Sepiapterin reductase (SPR) and dihydrofolate reductase (DHFR) are crucial enzymes in the BH4 de novo synthesis pathway and salvage pathway, respectively. Dihydrobiopterin (BH2) is an oxidized product of BH4 metabolism. The ratio of BH4/BH2 is a key indicator of the stability of BH4 levels. The de novo pathway of BH4 synthesis is well-defined; however, little is known about the mechanisms of the salvage pathway in insects. Herein, we used the natural BmSPR mutant silkworm (lem) as a resource material. Our results reveal that the BmDHFR expression and the BH4/BH2 ratio were remarkably higher in lem as compared to the wild-type silkworm. In BmN cells, knockdown of BmSpr showed increased BmDHFR expression, while the BH4/BH2 ratio decreased after BmDhfr knockdown by RNAi. Furthermore, simultaneous RNAi of BmSpr and BmDhfr showed a further decrease in the BH4/BH2 ratio. These manifest that the expression of BmDHFR is up-regulated to trigger an increase in the BH4/BH2 ratio when the de novo synthesis of BH4 is blocked in silkworm. Additionally, the knockdown of BmSpr in wild-type silkworms also showed an increased BmDHFR level and BH4/BH2 ratio. Taken together, when the silkworm BH4 de novo synthesis pathway is blocked, the salvage pathway is activated, and BmDHFR plays an important role in maintaining the metabolic balance of silkworm BH4. This study enriches our understanding of the molecular mechanism of the BH4 salvage pathway and lays a good foundation for further studies on BH4 using the silkworm as a model insect. [Display omitted] • The down-regulation of BmSpr leads to the up-regulation of BmDHFR in Bombyx mori. • BmDHFR plays a significant role in maintaining BH4 level in lem mutants. • Elevated SP content causes a yellow body color of the silkworm. • The increase of BmDHFR expression causes the up-regulation of BH4/BH2 ratio. [ABSTRACT FROM AUTHOR]

As a major public health problem, the prevalence of Acinetobacter baumannii (A. baumannii) infections in hospitals due to the pathogen's multiple-antibiotic resistance has attracted extensive attention. We previously reported a series of 1,3-diamino-7H-pyrrolo[3,2-f]quinazoline (PQZ) compounds, which were designed by targeting Escherichia coli dihydrofolate reductase (ecDHFR), and exhibited potent antibacterial activities. In the current study, based on our molecular-modeling study, it was proposed that PQZ compounds may function as potent A. baumannii DHFR (abDHFR)-inhibitors as well, which inspired us to consider their anti-A. baumannii abilities. We further found that three PQZ compounds, OYYF-171, -172, and -175, showed significant antibacterial activities against A. baumannii, including multidrug-resistant (MDR) strains, which are significantly stronger than the typical DHFR-inhibitor, trimethoprim (TMP), and superior to, or comparable to, the other tested antibacterial agents belonging to β-lactam, aminoglycoside, and quinolone. The significant synergistic effect between the representative compound OYYF-171 and the dihydropteroate synthase (DHPS)-inhibitor sulfamethoxazole (SMZ) was observed in both the microdilution-checkerboard assay and time-killing assay, which indicated that using SMZ in combination with PQZ compounds could help to reduce the required dosage and forestall resistance. Our study shows that PQZ is a promising scaffold for the further development of folate-metabolism inhibitors against MDR A. baumannii. [ABSTRACT FROM AUTHOR]

Background: Talaromyces marneffei (formerly Penicillium marneffei) is an important thermally dimorphic fungus endemic which is characterized by one of the most frequent opportunistic infections in HIV/AIDS patients, mainly prevalent in Southeast Asia, southern China, and northeastern India. Cotrimoxazole(CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi, thereby commonly used to prevent several opportunistic infections among HIV/AIDS patients. In addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis are considered to have the potential to prevent T. marneffei infection in HIV/AIDS patients receiving antiretroviral therapy (ART). However, the effect of cotrimoxazole towards T. marneffei fungus in vitro remains unclear. Methods: Human THP-1 macrophages were used as cell model in vitro to explore the effect and mechanism of cotrimoxazole resistance towards T. marneffei. Cell viability assay and drug sensitivity colony forming units (CFU) experiments were conducted to determine the minimum inhibitory concentration (MIC) of cotrimoxazole inside and outside THP-1 macrophages respectively. Enzyme-linked immunosorbent assay (Elisa) was used to measure the concentration of Dihydropteroic acid synthetase (DHPS), Dihydrofolate synthetase (DHFS) and Dihydrofolate reductase (DHFR) between T. marneffei adding TMP/SMX and without adding TMP/SMX group respectively. Real-time fluorescence quantitative PCR(qPCR) was performed to detect the mRNA expression levels in Dectin-1 mediated signaling pathway and downstream inflammatory cytokines including IL-6, IL-10, IL-23A, CXCL8 and TNF-α released by T. marneffei-infected macrophages between adding TMP/SMX and without adding TMP/SMX group respectively. Results: Cotrimoxazole can inhibit the proliferation of T. marneffei within safe concentration inside and outside THP-1 macrophages. Drug susceptibility results showed the minimal inhibit concentration(MIC) of 1:5 TMP/SMX was ranging from 14/70 to 68/340 μg/ml. The MIC of SMX was ranging from 100 to 360 μg/ml. The MIC of TMP was ranging from 240 to 400 μg/ml outside macrophages. The MIC of TMP/SMX was ranging from 36/180 to 68/340 μg/ml. The MIC of SMX was ranging from 340 to 360 μg/ml. The MIC of TMP was ranging from 320 to 400 μg/ml inside macrophages. The synergistic interaction of 1:5 TMP/SMX was more effective in inhibiting T. marneffei than separate SMX and TMP. DHPS, DHFS and DHFR can be inhibited by cotrimoxazole within safe and effective concentration. Dectin-1 expression is increased following T. marneffei infection, leading to the increase of IL-6, IL-10, IL-23A and the decrease of CXCL8 and TNF-α. Conversely, cotrimoxazole decrease the levels of Dectin-1, IL-6, IL-10, IL-23A and increase the levels of CXCL8 and TNF-α, thereby enhancing the intracellular killing-T. marneffei capacity of macrophages. Conclusions: Our findings indicated that cotrimoxazole directly inhibited T. marneffei growth by blocking DHPS, DHFS and DHFR and indirectly inhibited T. marneffei growth perhaps by regulating the Dectin-1 signaling pathway, which may effectively interfere with the defense ability of the host against T. marneffei infection. [ABSTRACT FROM AUTHOR]

In response to antibiotics that inhibit a bacterial enzyme, resistance mutations inevitably arise. Predicting them ahead of time would aid target selection and drug design. The simplest resistance mechanism would be to reduce antibiotic binding without sacrificing too much substrate binding. The property that reflects this is the enzyme “vitality”, defined here as the difference between the inhibitor and substrate binding free energies. To predict such mutations, we borrow methodology from computational protein design. We use a Monte Carlo exploration of mutation space and vitality changes, allowing us to rank thousands of mutations and identify ones that might provide resistance through the simple mechanism considered. As an illustration, we chose dihydrofolate reductase, an essential enzyme targeted by several antibiotics. We simulated its complexes with the inhibitor trimethoprim and the substrate dihydrofolate. 20 active site positions were mutated, or “redesigned” individually, then in pairs or quartets. We computed the resulting binding free energy and vitality changes. Out of seven known resistance mutations involving active site positions, five were correctly recovered. Ten positions exhibited mutations with significant predicted vitality gains. Direct couplings between designed positions were predicted to be small, which reduces the combinatorial complexity of the mutation space to be explored. It also suggests that over the course of evolution, resistance mutations involving several positions do not need the underlying point mutations to arise all at once: they can appear and become fixed one after the other.

Endothelial malfunction is a major contributor to early or delayed vasospasm after subarachnoid hemorrhage (SAH). As a representative form of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling leads to a reduction in nitric oxide (NO) generated by endothelial cells. In this study, we investigated how the interaction between endothelial NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and DHFR (dihydrofolate reductase) contributes to eNOS uncoupling after SAH. Setanaxib and the adeno-associated virus (AAV) targeting brain vascular endothelia were injected through the tail vein and the expression and localization of proteins were examined by western blot and immunofluorescence staining. The NO content was measured using the NO assay kit, and laser speckle contrast imaging was used to assess cortical perfusion. ROS (reactive oxygen species) level was detected by DHE (dihydroethidium) staining, DCFH-DA (2′,7′-dichlorofluorescin diacetate) staining and H 2 O 2 (hydrogen peroxide) measurement. The Garcia score was employed to examine neurological function. Setanaxib is widely used for its preferential inhibition for NOX1/4 over other NOX isoforms. After endothelial NOX4 was inhibited by Setanaxib in a mouse model of SAH, the endothelial DHFR level was significantly elevated, which attenuated eNOS uncoupling, increased cortical perfusion, and improved the neurological function. The protective role of inhibiting endothelial NOX4, however, disappeared after knocking down endothelial DHFR. Our results suggest that endothelial DHFR decreased significantly because of the elevated level of endothelial NOX4, which aggravated eNOS uncoupling after SAH, leading to decreased cortical perfusion and worse neurological outcome. [Display omitted] • Endothelial NOX4 increased after SAH, along with decreased DHFR and NO content. • Elevated endothelial NOX4 contributed to eNOS uncoupling after SAH. • Inhibition of endothelial NOX4 improved cortical perfusion after SAH. • Decreased cortical perfusion could be alleviated via attenuating eNOS uncoupling. • Protective role of Setanaxib was reversed after knocking down endothelial DHFR. [ABSTRACT FROM AUTHOR]

Pathophysiology associated with Huntington’s disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD. [ABSTRACT FROM AUTHOR]

In this paper we describe the synthesis of new N-heterocyclic carbene (NHC) gold(I) derivatives with flavone-derived ligands with a propargyl ether group. The compounds were screened for their antimicrobial and anticancer activities, showing greater activity against bacteria than against colon cancer cells (Caco-2). Complexes [Au(L2b)(IMe)] (1b) and [Au(L2b)(IPr)] (2b) were found to be active against both Gram-positive and Gram-negative strains. The mechanism of action of 1b was evaluated by measurement of thioredoxin reductase (TrxR) and dihydrofolate reductase (DHFR) activity, besides scanning electron microscopy (SEM). Inhibition of the enzyme thioredoxin reductase is not observed in either Escherichia Coli or Caco-2 cells; however, DHFR activity is compromised after incubation of E. coli cells with complex 1b. Moreover, loss of structural integrity and change in bacterial shape is observed in the images obtained from scanning electron microscopy (SEM) after treatment E. coli cells with complex 1b. [ABSTRACT FROM AUTHOR]

Aims: 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. Methods and Results: 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. Conclusion: These findings suggest that glabridin represents the prototypical member of an exciting structural class of natural antimicrobial agents. Significance and Impact of the Study: This study reports a novel mechanism of bactericidal activity of glabridin against Staph. aureus. [ABSTRACT FROM AUTHOR]

ABSTRACT Since 2010, the human-infecting malaria parasite Plasmodium ovale spp. has been divided into two genetically distinct species, P. ovale wallikeri and P. ovale curtisi. In recent years, application of whole-genome sequencing (WGS) to P. ovale spp. allowed to get a better understanding of its evolutionary history and discover some specific genetic patterns. Nevertheless, WGS data from P. ovale spp. are still scarce due to several drawbacks, including a high level of human DNA contamination in blood samples, infections with commonly low parasite density, and the lack of robust in vitro culture. Here, we developed two selective whole-genome amplification (sWGA) protocols that were tested on six P. ovale wallikeri and five P. ovale curtisi mono-infection clinical samples. Blood leukodepletion by a cellulose-based filtration was used as the gold standard for intraspecies comparative genomics with sWGA. We also demonstrated the importance of genomic DNA preincubation with the endonuclease McrBC to optimize P. ovale spp. sWGA. We obtained high-quality WGS data with more than 80% of the genome covered by ≥5 reads for each sample and identified more than 5,000 unique single-nucleotide polymorphisms (SNPs) per species. We also identified some amino acid changes in pocdhfr and powdhfr for which similar mutations in P. falciparum and P. vivax are associated with pyrimethamine or cycloguanil resistance. In conclusion, we developed two sWGA protocols for P. ovale spp. WGS that will help to design much-needed large-scale P. ovale spp. population studies. IMPORTANCE Plasmodium ovale spp. has the ability to cause relapse, defined as recurring asexual parasitemia originating from liver-dormant forms. Whole-genome sequencing (WGS) data are of importance to identify putative molecular markers associated with relapse or other virulence mechanisms. Due to low parasitemia encountered in P. ovale spp. infections and no in vitro culture available, WGS of P. ovale spp. is challenging. Blood leukodepletion by filtration has been used, but no technique exists yet to increase the quantity of parasite DNA over human DNA when starting from genomic DNA extracted from whole blood. Here, we demonstrated that selective whole-genome amplification (sWGA) is an easy-to-use protocol to obtain high-quality WGS data for both P. ovale spp. species from unprocessed blood samples. The new method will facilitate P. ovale spp. population genomic studies.

Background: Alzheimer’s disease (AD) is a type of disease frequently occurs in the elderly population. Diagnosis and treatment methods for this disease are still lacking, and more research is required. In addition, little is known about the function of the circular RNAs (circRNAs) in AD. Methods: In this research, RNA expression data of AD from the Gene Expression Omnibus (GEO) database were downloaded. The expression levels of circRNAs in cerebrospinal fluid samples of healthy participants and AD patients were measured by reverse transcription‑quantitative PCR (RT-qPCR). The diagnosed value of differential expressed circRNAs was analyzed with the Receiver operating characteristic curve (ROC). Pathways related to circ_0001535 were found using gene set enrichment analysis (GSEA) and Metascape. The direct interactions between circ_0001535 and E2F transcription factor 1 (E2F1) or E2F1 and dihydrofolate reductase (DHFR) were verified using Chromatin immunoprecipitation (ChIP) and RNA Binding Protein Immunoprecipitation (RIP) assays. Cell Counting Kit-8 (CCK8) and flow cytometry were used to identify the function of circ_0001535/E2F1/DHFR axis on the proliferation and apoptosis of AD cells. Results: In total, 12 circRNAs have been linked to AD diagnosis. The expression levels of 7 circRNAs differed between AD patients and control groups. Circ_0001535 had the most diagnose value among these circRNAs. Hence, circ_0001535 was regarded as a key circRNA in the present study. E2F1/DHFR axis was predicted to be regulated by circ_0001535. In addition, IP assays experiment results showed that E2F1 could bind to the promoter region of DHFR and be regulated by circ_0001535. In vitro results showed that circ_0001535 overexpression could promote DHFR expression, while E2F1 knock down could inhibit DHFR expression in SH-SY5Y cells. Finally, rescue experiments suggested that circ_0001535 could reduce Aβ25-35-induced SH-SY5Y cell proliferation and facilitate apoptosis through E2F1/DHFR axis. Conclusions: Our research in AD circRNA can offer important information regarding the role of specific circRNAs in the AD environment and point to specific future areas of therapeutic intervention in AD.

We compared the binding properties and dynamics of three experimentally reviewed isoforms of human dihydrofolate reductase (DHFR). The cytoplasmic variants including isoforms1 and 2 (iso1 and iso2) are produced by alternative splicing; while the mitochondrial form is located in the mitochondria. The iso1 as the canonical sequence contains 187 residues, and iso2 differs from the iso1, where it has 1–52 residues missing at the N-terminus of canonical sequence. Here, the structural models of the iso2 and mitochondrial forms were constructed by the MODELLER program using the crystal structure of the iso1 as the template. Bioinformatics analysis on ligand-bearing structures demonstrates that mitochondrial variant forms more stable complex with ligands compared with iso1 and 2, indicating their different binding properties. The root mean square fluctuation (RMSF) data suggest that C-terminus of iso1 contains two representative highly flexible fragments, while iso2 contains a highly flexible fragment at N-terminus end. Interestingly, both ends of mitochondrial variant have a degree of rigidity. Finally, the observation of differences in structural dynamics and binding properties predicts that the simultaneous existence of enzyme isoforms is a way to increase the speed of the enzyme maneuver in response to various environmental conditions. This prediction needs to be tested experimentally. [ABSTRACT FROM AUTHOR]

Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H4B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling.

This article includes a general overview of the published research on a topic relevant to biomedical sciences research, pharma-industries and healthcare sector. We have presented a concise information on three enzymes. These biomolecules have been investigated for their biocatalytic activities beneficial in the detection of drugs and their metabolites present in micro-quantities in samples of blood, urine, and other body fluids, such as salicylate hydroxylase, and dihydrofolate reductase. Some enzymes are useful in biotransformation of compounds to convert them in an optically active form, such as lipase. The information presented in this article has been collected from the published studies on their catalytic function, and biosynthesis using selected microorganisms. Several diagnostic assays are currently using enzymes as effective biocatalysts to perform the detection-test. For the marketing and consumer's convenience, pharmaceutical companies have designed biosensors and diagnostic kits by incorporating specific enzymes for rapid tests required in pathology, as well as for the quantification of certain metabolites and chemicals in pathology samples in a shorter time. For such purpose use of enzymes synthesized by selected specific microorganisms is economical.

Aims Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. Methods and results Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5′-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe −/− (Apoe −/− Gch1 fl/fl Tie2 Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe −/− Gch1 fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si- GCH1- or si- DHFR -transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe −/− mice through increasing DHFR and salvaging BH4 biosynthesis. Conclusion The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability. [ABSTRACT FROM AUTHOR]

Drug resistance is an important problem hindering malaria elimination in tropical areas. Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes confer resistance to antifolate drug, sulfadoxine-pyrimethamine (SP) while P. falciparum chloroquine-resistant transporter (Pfcrt) genes caused resistance to chloroquine (CQ). Decline in Pfdhfr/Pfdhps and Pfcrt mutations after withdrawal of SP and CQ has been reported. The aim of present study was to investigate the prevalence of Pfdhfr, Pfdhps, and Pfcrt mutation from 2 endemic areas of Thailand. All of 200 blood samples collected from western area (Thai-Myanmar) and southern area (Thai-Malaysian) contained multiple mutations in Pfdhfr and Pfdhps genes. The most prevalent haplotypes for Pfdhfr and Pfdhps were quadruple and double mutations, respectively. The quadruple and triple mutations of Pfdhfr and Pfdhps were common in western samples, whereas low frequency of triple and double mutations was found in southern samples, respectively. The Pfcrt 76T mutation was present in all samples examined. Malaria isolated from 2 different endemic regions of Thailand had high mutation rates in the Pfdhfr, Pfdhps, and Pfcrt genes. These findings highlighted the fixation of mutant alleles causing resistance of SP and CQ in this area. It is necessary to monitor the re-emergence of SP and CQ sensitive parasites in this area. [ABSTRACT FROM AUTHOR]

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 μM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa.

Aim. Search for new antibacterial agents with dihydrofolate reductase-inhibitory activity among N-(R-carbamothiol)cycloalkylcarboxamides using in silico and in vitro methodology, SAR analysis to optimize the synthesis of new potential antinicrobials. Methods. Molecular docking, in vitro DHFR inhibition assay, antimicrobial evaluation, SAR analysis, statistical methods. Results. According to the results of molecular docking to the active center of dihydrofolate reductase (DHFR), namely affinity, the main types of interactions and arrangement in the active center of the enzyme, several N-(R-carbamothioyl)cycloalkylcarboxamides were selected for their inhibitory effect. Based on in vitro screening, few promising compounds with high ability to inhibit DHFR were identified. It was found, that diacylsemicarbazides are more effective inhibitors of DHFR compared to acylthioureas. The studies on antibacterial activity have revealed several promising compounds, namely N-(2-R-hydrazine-1-carbonothioyl)cycloalkanecarboxamides, as highly active antimicrobial agents against E. coli and St. aureus (MIC 3.125-25.0 µg/ml) with high DHFR-inhibitory effect, the activity of which competes with the comparison drug "Nitrofurazone". This justifies the continuation of systematic research in this direction. Conclusions. A well-founded search among N-(R-carbamothiol)cycloalkylcarboxamides for new antibacterial agents with dihydrofolate reductase-inhibitory activity, using in silico and in vitro methodology, established relationship between the chemical structure and activity aimed at further design of new potential drug agents. [ABSTRACT FROM AUTHOR]

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 μM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to characterize molecular mechanisms of resistance to trimethoprim and other antibiotics in Streptococcus pneumoniae isolates from HIV-infected adults in Dar es Salaam, Tanzania. Methods: A total of 1877 nasopharyngeal swabs were collected and screened for pneumococcal colonization from 537 newly diagnosed individuals with HIV at four clinic visits during a 1-year follow-up from 2017–2018 as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov ID: NCT03087890). Results: A total of 76 pneumococcal isolates were obtained. Of the 70 isolates that could be serotyped, 42 (60.0%) were vaccine serotypes included in pneumococcal conjugate vaccine 23 (PCV23). The majority of isolates (73.7%; 56/76) were non-susceptible to penicillin (MICs of 0.06–2 μg/mL). Isolates were frequently resistant to co-trimoxazole (trimethoprim/sulfamethoxazole) (71.1%) but less so to azithromycin (22.4%), erythromycin (21.1%), chloramphenicol (18.4%), tetracycline (14.5%), clindamycin (10.5%) and levofloxacin (0%). Moreover, 26.3% were multidrug-resistant (resistant to ≥3 antibiotic classes). Vaccine-type pneumococci were resistant to more classes of antibiotics, were more frequently resistant to erythromycin, azithromycin, clindamycin and tetracycline, and had higher MICs to penicillin (median, 0.19 μg/mL; range, 0.002–1.5 μg/mL) compared with non-vaccine serotypes (median, 0.125 μg/mL; range, 0.012–0.25 μg/mL) (P = 0.003). Co-trimoxazole-resistant isolates carried from 1 to 11 different mutations in the dihydrofolate reductase (DHFR) gene, most commonly Ile100Leu (100%), Glu20Asp (91.8%), Glu94Asp (61.2%), Leu135Phe (57.1%), His26Tyr (53.1%), Asp92Ala (53.1%) and His120Gln (53.1%). Conclusion: Streptococcus pneumoniae isolated from HIV-diagnosed patients were frequently non-susceptible to penicillin and co-trimoxazole. Most isolates carried multiple mutations in DHFR.