Dihydrofolate Reductase Protein Research Articles

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo.

AbstractThere is enormous interest in strategies to efficiently traffic biologics–proteins, nucleic acids, and complexes thereof–into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic knowledge regarding the bioinorganic chemistry of zinc(II) coordination in small proteins, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity upon arrival in the cytosol. Delivery of the pH-sensitive protein dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins.Abstract Figure

Unveiling the Connection: Viral Infections and Genes in dNTP Metabolism.

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.

Anti-colorectal Cancer Activity of Quinazoline Derivatives: A Comprehensive Review

Introduction and Objective: The identification of a bioactive template (or lead) is one of the important features of modern drug discovery. Natural products, synthetic and biotechnological sources, serve as important templates for the development of novel bioactive molecules. Quinazoline is one of the heterocyclic templates present in many natural and synthetic drugs and exhibits various biological activities, including anticancer, by blocking the pharmacological pathway of different targets. Methodology: In this study, the data was collected from the literature and patents to examine the anticolorectal cancer efficacy of quinazoline compounds and their mechanism of actions. According to the published literature and patents, the benzene and/or pyrimidine rings of the quinazoline have been substituted with amino groups or substituted amino groups to develop novel analogues endowed with anticancer properties. The anti-colorectal cancer activity of quinazolines was due to the flexible chain containing terminal phenyl and/or heterocyclic rings (thiazole, pyrazole, piperidine, piperazine, etc.). Results: These quinazoline derivatives were found to inhibit the growth of colorectal cancer cells by modulating the expression of specific genes and proteins involved in cancer progression, such as receptor tyrosine kinases, epidermal growth factor receptors, dihydrofolate reductase, topoisomerases, histone deacetylase, and apoptotic proteins. Conclusion: These findings suggest that the quinazoline nucleus may be exploited to identify new anticolorectal cancer agents with suitable pharmacokinetic profiles.

Bioactive compounds extracted from leaves of G. cyanocarpa using various solvents in chromatographic separation showed anti-cancer and anti-microbial potentiality in in silico approach

The study was carried out to determine the possible bioactive compounds from Ethanol, Methanol, Petroleum ether, and Dichloromethane fractions of Glycosmic cyanocarpa (G. cyanocarpa). Analysis of these extracts was performed using a mass spectrometer detector installed with gas chromatography (GC–MS/MS) utilizing a method named electron impact ionization (EI). The mass spectrum of each extract was compared against the information incorporated in the library (NIST and Wiley) which provides the chemical structure with the name and molecular masses of the identified compounds. A total of 73 compounds (25 from ethanol, 19 from methanol and 5 from dichloromethane (DCM), and 24 from petroleum ether fractions) were identified from various fractions of the plants. Fourier Transform infrared spectroscopy (FTIR) analysis of the crude extracts was done to identify the functional groups of the plant derived compounds. The isolated compounds were subjected to comparison with the standard drugs towards the active binding sites of epidermal growth factor receptor (EGFR) and dihydrofolate reductase (DHFR) proteins for further evaluation of their cytotoxic and antimicrobial activity, respectively. Stigmasterol, beta-Sitosterol, Pyrazol-5-amine, 3-methyl-1,4-diphenyl-, 1,2-Benzenediol,4-(2-aminopropyl)-18,19-Secoyohimban-19-oic acid, and Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) showed maximum binding affinity towards EGFR and 16,17,20,21-tetradehydro-16-(hydroxymethyl)-, methyl ester, (15 beta.,16E)-, Stigmasterol, beta-Sitosterol, Pyrazol-5-amine, 3-methyl-1,4-diphenyl- revealed highest binding affinity towards DHFR receptor. According to the current research, G. cyanocarpa may be a useful natural source for controlling antibacterial and anticancer activity. For thorough phytochemical screening and determining precise mechanisms of action, additional research is required.

Methotrexate-based PROTACs as DHFR-specific chemical probes

Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.

Elongation Factor P Modulates the Incorporation of Structurally Diverse Noncanonical Amino Acids into Escherichia coli Dihydrofolate Reductase.

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Phytochemical Profiling and Pharmacological Evaluation of Leaf Extracts of Ruellia tuberosa L.: An In Vitro and In Silico Approach.

The present study was designed to appraise the photoprotective, antioxidant, and antibacterial bioactivities of Ruellia tuberosa leaves extracts (RtPE, RtChl, RtEA, RtAc, RtMe, and RtHMe). The results showed that, RtHMe extracts of R. tuberosa was rich in total phenolic content, i. e., 1.60 mgGAE/g dry extract, while highest total flavonoid content was found in RtAc extract, i. e., 0.40 mgQE/g. RtMe showed effective antioxidant activity (%RSA: 58.16) at the concentration of 120 μL. RtMe, RtEA and RtHMe exhibited effective in vitro antibacterial activity against Gram-negative bacteria (E. coli). In silico docking studies revealed that paucifloside (-11.743 kcal/mol), indole-3-carboxaldehyde (-7.519 kcal/mol), nuomioside (-7.275 kcal/mol), isocassifolioside (-6.992 kcal/mol) showed best docking score against PDB ID 2EX8 [penicillin binding protein 4 (dacB) from Escherichia coli, complexed with penicillin-G], PDB ID 6CQA (E. coli dihydrofolate reductase protein complexed with inhibitor AMPQD), PDB ID 2Y2I [Penicillin-binding protein 1B in complex with an alkyl boronate (ZA3)] and PDB ID 2OLV (from S. aureus), respectively. Docked phytochemicals also showed good drug likeness properties.

Anticancer Activities of Seven Peronemins (A2, A3, B1, B2, B3, C1, and D1) from Peronema canescens Jack: A Prediction Studies

Cancer is one of the leading causes of human death. In 2019, it was reported that cancer was the second (22%) cause of death due to non-communicable diseases in the world's population. Research for alternative anticancer drugs is still being done, including anticancer from plants. One of the plants that have the potential to be developed as an anticancer alternative is the sungkai plant. Sungkai leaves contain many bioactive compounds, one of which is the clerodane-type diterpenoids, peronemins, A2 (1), A3 (2), B1 (3), B2 (4), B3 (5), C1 (6), and D1 (7). The aim of this study was to initial screen the potential of seven Peronemins compounds in Sungkai leaves extract as anticancer candidates. Initial screening was carried out by predicting in-silico anticancer activity of the seven compounds. Dihydrofolate reductase inhibitor (DHFR inhibitor) is one of the anticancer activity screening approaches. DHFR Inhibitor activity from perenomins derivatives with pIC50 values ​​of 0.785 (A2), respectively; 0.785 (A3); 0.799 (B2); 0.799 (B3); 0.799 (C1 and D1). In addition, from compounds 1,2,3,4,5 peronemin derivatives have potential anticancer activity through interaction with the target protein Voltage-gated potassium channel subunit while compounds 6, 7 also have biological activity potential anticancer on target protein Dihydrofolate reductase.

Screening potential inhibitor from actinomycetes for Dihydrofolate reductase of Staphylococcus aureus – In vitro and in silico studies

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.

Is Posttranslational Folding More Efficient Than Refolding from a Denatured State: A Computational Study.

The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular machine responsible for protein synthesis, is known to interact with nascent proteins, adding further complexity to the protein folding landscape. Consequently, it is unclear whether the folding pathways of proteins are conserved on and off the ribosome. The main question remains: to what extent does the ribosome help proteins fold? To address this question, we used coarse-grained molecular dynamics simulations to compare the mechanisms by which the proteins dihydrofolate reductase, type III chloramphenicol acetyltransferase, and d-alanine-d-alanine ligase B fold during and after vectorial synthesis on the ribosome to folding from the full-length unfolded state in bulk solution. Our results reveal that the influence of the ribosome on protein folding mechanisms varies depending on the size and complexity of the protein. Specifically, for a small protein with a simple fold, the ribosome facilitates efficient folding by helping the nascent protein avoid misfolded conformations. However, for larger and more complex proteins, the ribosome does not promote folding and may contribute to the formation of intermediate misfolded states cotranslationally. These misfolded states persist posttranslationally and do not convert to the native state during the 6 μs runtime of our coarse-grain simulations. Overall, our study highlights the complex interplay between the ribosome and protein folding and provides insight into the mechanisms of protein folding on and off the ribosome.

Computational screening of FDA-approved drugs to identify potential TgDHFR, TgPRS, and TgCDPK1 proteins inhibitors against Toxoplasma gondii

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world, because about a third of the world's population is seropositive for toxoplasmosis. Treatment regimens for toxoplasmosis have remained unchanged for the past 20 years, and no new drugs have been introduced to the market recently. This study, performed molecular docking to identify interactions of FDA-approved drugs with essential residues in the active site of proteins of T. gondii Dihydrofolate Reductase (TgDHFR), Prolyl-tRNA Synthetase (TgPRS), and Calcium-Dependent Protein Kinase 1 (TgCDPK1). Each protein was docked with 2100 FDA-approved drugs using AutoDock Vina. Also, the Pharmit software was used to generate pharmacophore models based on the TgDHFR complexed with TRC-2533, TgPRS in complex with halofuginone, and TgCDPK1 in complex with a bumped kinase inhibitor, RM-1–132. Molecular dynamics (MD) simulation was also performed for 100 ns to verify the stability of interaction in drug–protein complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis evaluated the binding energy of selected complexes. Ezetimibe, Raloxifene, Sulfasalazine, Triamterene, and Zafirlukast drugs against the TgDHFR protein, Cromolyn, Cefexim, and Lactulose drugs against the TgPRS protein, and Pentaprazole, Betamethasone, and Bromocriptine drugs against TgCDPK1 protein showed the best results. These drugs had the lowest energy-based docking scores and also stable interactions based on MD analyses with TgDHFR, TgPRS, and TgCDPK1 drug targets that can be introduced as possible drugs for laboratory investigations to treat T. gondii parasite infection.

Synthesis, Characterization, Bioactivity Screening and Computational Studies of Diphenyl−malonohydrazides and Pyridines Derivatives

AbstractA series of new hydrazide (3 a–j) and pyridine (11 a–j) derivatives were synthesized using a convergent synthetic methodology by condensation of malono‐di(2‐phenylhydrazide) with arylidene malononitrile or arylidene ethyl cyanoacetate derivatives. The synthesized compounds (3, 11 a–j) were characterized using via IR, 1H‐, 13C‐NMR, and MS spectroscopies as well as elemental analysis. The biological activity of these molecules has been evaluated in vitro against two gram‐positive bacteria (Staphylococcus aureus and Streptococcus pneumoniae) and one‐gram negative bacteria (Escherichia coli), as well as one fungus (Candida albicans). The results of the bioactive assay revealed that the synthesized pyridine (11 a–j) derivatives had greater antibacterial efficacy than the hydrazide (3 a–j) derivatives and were comparable to the reference drug Augmentin. Furthermore, docking studies against the Staphylococcus aureus dihydrofolate reductase (DHFR) protein revealed that pyridine derivatives (11 a–j) had higher binding interactions affinity (ΔG=−9.59∼−7.69 kcal/mol) than diphenyl−malonohydrazide derivatives (3 a–j), which achieved a binding affinity in the range of (ΔG=−9.65∼−6.77 kcal/mol), supporting the experimental results. Finally, DFT and TD‐DFT were used to gain a better understanding of the structure‐activity relationship and biological activity of the new synthesized hydrazide/pyridine derivatives.

Dry and wet lab experiences of certain curcumin analogues’ antibacterial effect on S.aureus

Curcumin analogues are widely used in numerous biological disciplines. They are widely employed in the pharmaceutical sector and offer good pharmacology application prospects in the present era. In the current study, two curcumin analogues—1,7-diphenylhepta-1,6-diene-3,5-dione (DPHDD) and 1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (BDMPHDD)—were tested in vitro against the bacterial strain S. aureus. The testing demonstrated that the ligands may have antibacterial potential. The rule of five was used to pre-filter the compounds' drug-like characteristics prior to computer analysis. Then, to determine the mechanism by which the compounds limit the growth of S. aureus, molecular docking research was carried out using the AutoDock 4.2 tool. Six distinct target proteins from S. aureus were chosen for this purpose (PDB ID: 1T2P, 3U2D, 2W9S, 1N67, 2ZCO, and 4H8E). The target protein Dihydrofolate reductase enzyme (PDB ID: 2W9S) and Staphylococcus aureus sortase-A (PDB ID: 1T2P) demonstrated a good binding affinity for the two analogues, (BDMPHDD) & (DPHDD) respectively.Due to the inactivation of these enzymes, the substances 1,7-diphenylhepta-1,6-diene-3,5-dione (DPHDD) and 1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (BDMPHDD) exhibit significant growth-inhibitory potential against S. aureus.

Biological Activity of Picrorhiza kurroa: A Source of Potential Antimicrobial Compounds against Yersinia enterocolitica.

Yersiniosis, caused by Yersinia enterocolitica, is the third most rampant zoonotic disease in Europe; the pathogen shows high antibiotic resistance. Herbs have multiple anti-microbial components that reduce microorganism resistance. Therefore, an extract of Picrorhiza kurroa (P. kurroa) was evaluated for potential antimicrobial activity. We report that the ethanolic extract of P. kurroa showed effective antimicrobial activity (zone of inhibition: 29.8 mm, Minimum inhibitory concentration (MIC): 2.45 mg/mL, minimum bactericidal concentration (MBC): 2.4 mg/mL) against Yersinia enterocolitica. Potential bioactive compounds from P. kurroa were identified using LC-MS, namely, cerberidol, annonidine A, benzyl formate, picroside-1, and furcatoside A. P. kurroa showed effective antimicrobial potential in skim milk at different pH, acidity, and water activity levels. P. kurroa affected the physiology of Yersinia enterocolitica and reduced the number of live cells. Yersinia enterocolitica, when incubated with P. kurroa extract, showed lower toxin production. Picroside-1 was isolated and showed higher antimicrobial potential in comparison to the standard antibiotic. Picroside-1 lysed the Yersinia enterocolitica cells, as observed under scanning electron microscopy. Docking revealed that picroside-1 (ligand) showed both hydrophilic and hydrophobic interactions with the dihydrofolate reductase (DHFR) protein of Yersinia enterocolitica and that DHFR is a possible drug target. The high activity and natural origin of Picroside-1 justify its potential as a possible drug candidate for Yersinia enterocolitica.

FtsH degrades dihydrofolate reductase by recognizing a partially folded species.

AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well‐characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N‐terminus or C‐terminus of the substrate. Results using peptide‐array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported.

Characterization of β-Lactamases and Multidrug Resistance Mechanisms in Enterobacterales from Hospital Effluents and Wastewater Treatment Plant.

Antimicrobials in wastewater promote the emergence of antibiotic resistance, facilitated by selective pressure and transfer of resistant genes. Enteric bacteria belonging to Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Citrobacter species (n = 126) from hospital effluents and proximate wastewater treatment plant were assayed for susceptibility to four antimicrobial classes. The β-lactamase encoding genes harbored in plasmids were genotyped and the plasmids were sequenced. A multidrug resistance phenotype was found in 72% (n = 58) of E. coli isolates, 70% (n = 43) of Klebsiella species isolates, and 40% (n = 25) of Enterobacter and Citrobacter species. Moreover, 86% (n = 50) of E. coli, 77% (n = 33) of Klebsiella species, and 25% (n = 4) of Citrobacter species isolates phenotypically expressed extended spectrum β-lactamase. Regarding ESBL genes, blaCTX-M-27 and blaTEM-1 were found in E. coli, while Klebsiella species harbored blaCTX-M-15, blaCTX-M-30, or blaSHV-12. Genes coding for aminoglycoside modifying enzymes, adenylyltransferases (aadA1, aadA5), phosphotransferases (aph(6)-1d, aph(3″)-Ib), acetyltransferases (aac(3)-IIa), (aac(6)-Ib), sulfonamide/trimethoprim resistant dihydropteroate synthase (sul), dihydrofolate reductase (dfrA), and quinolone resistance protein (qnrB1) were also identified. Monitoring wastewater from human sources for acquired resistance in clinically important bacteria may provide a cheaper alternative in regions facing challenges that limit clinical surveillance.

Design, synthesis, antimicrobial evaluations and in silico studies of novel pyrazol-5(4H)-one and 1H-pyrazol-5-ol derivatives

A new series of 1,4-disubstituted 3-methylpyrazol-5(4 H )-one derivatives were synthesized by reacting various substituted aromatic aldehydes with 3-methylpyrazol-5(4 H )-one derivatives through Knoevenagel condensation by conventional as well as by exposure to microwave irradiations. After that newly synthesized compounds of 1,4-disubstituted 3-methyl-1 H -pyrazol-5-ol were prepared from these derivatives by reduction reaction of sodium borohydride at 0–5 °C. Sixty-four heterocyclic compounds containing a pyrazole moiety were synthesized with good to excellent yields (51 to 91%). Compounds ( 3d , 3m , 4a , 4b , 4d , and 4g ) showed potent antibacterial activity against MSSA (Methicillin-susceptible strains of Staphylococcus aureus ) and MRSA (Methicillin-resistant strains of Staphylococcus aureus ) with MIC (the minimum inhibitory concentration) ranging between 4 and 16 µg/mL as compared to ciprofloxacin (MIC = 8–16 µg/mL). Compounds ( 4a , 4h , 4i , and 4l ) showed potent antifungal activity against Aspergillus niger with MIC ranging between 16 and 32 µg/mL as compared to fluconazole (MIC = 128 µg/mL). In particular, compound 4a exhibited the strongest activity among the synthesized compounds in both bacterial and fungal strains with MIC ranging between 4 and 16 µg/mL. Furthermore, the nine most active compounds showed a good ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile in comparison to ciprofloxacin and fluconazole as reference drugs. Molecular docking predicted that DHFR (dihydrofolate reductase) protein from Staphylococcus aureus and NMT ( N -myristoyl transferase) protein from Candida albicans are the most suitable targets for the antimicrobial activities of these potent compounds.

Ethanol and Acetic Acid Induces Conformational Changes in Zebrafish Dihydrofolate Reductase Protein

An equilibrium unfolding research of the recombinant zDHFR would provide information on the conformational changes of protein. In present work, the conformation of recombinant zDHFR was investigated in presence of ethanol and acetic acid. Equilibrium unfolding of recombinant zDHFR was monitored by enzymatic assay after denaturation by ethanol and acetic acid at 340 nm. Changes in secondary and tertiary structure of zDHFR with increasing ethanol and acetic acid concentrations were investigated in far-UV circular dichroism (CD) (190 to 250 nm) and fluorescence spectroscopy (emission spectra from 300 to 400 nm with an excitation wavelength of 295 nm) methods. It has been observed that in case of acetic acid-induced denaturation of zDHFR, the shift from native to denatured state occurs in a single step, whereas intermediates or non-native states are found at low concentrations of ethanol. The recent findings have significant implications for understanding how ethanol and acetic acid affect protein structure.

Establishment of a Dihydrofolate Reductase Gene Knock-In Zebrafish Strain to Aid Preliminary Analysis of Congenital Heart Disease Mechanisms.

Background: The dihydrofolate reductase (DHFR) gene is imperative in development, therefore it is essential to explore its effects on heart development. Thus, here a dhfr zebrafish knock-in (KI) strain was constructed.Methods: CRISPR/Cas9 technology was used to establish the dhfr KI zebrafish strain. This strain was hybridized with TgG fluorescent strain zebrafish to observe the phenotypes of heart shape, size, and circularization direction. Wild-type (WT) and KI zebrafish were then dissected and histologically stained to observe pathological changes. Western blot analysis was used to verify the increased expressions of zebrafish genes after KI. Hybridization experiments were used to confirm the presence of abnormal gonadal dysplasia.Results: The zebrafish dhfr KI strain was successfully constructed through CRISPR/Cas9 technology. At 6 days post fertilization (dpf), microscopic examinations of KI (homozygous) specimens revealed pericardial effusions, heart compressions, and curled tails. Compared with WT, the Hematoxylin and Eosin (H&E) tissue sections of KI-homozygous zebrafish showed defects such as reduced atria and ventricles. Western blot analysis indicated that the expression of the DHFR protein increased in both heterozygotes and homozygotes of dhfr KI zebrafish. Hybridization experiments revealed that dhfr KI may affect gonadal function.Conclusion: The DHFR gene plays an important regulatory role in the process of heart development, and copy number variations (CNVs) of this gene may constitute a new pathogenic mechanism of congenital heart disease (CHD).

Construction of 3D Model of Protein Drug Targets for Renibacterium Salmoninarum - A Bacterial Pathogen Causing Bacterial Kidney Disease in Young Salmonid Fish

The aim of this study is to construct 3D models of potential drug targets for the Bacterial Kidney Disease (BKD) causing pathogen Renibacterium salmoninarum. The bacterial pathogen Renibacterium salmoninarum was selected for homology modeling studies since there were no known protein structures of the organism reported in the NCBI database. The reported protein sequences were run through DrugBank to pick out drug-targets. Online databases and web tools such as PMDB, UniProt, Drug Bank, and SwissModel were employed in this analysis. An aggregate of 412 protein sequences were identified as potential drug targets and were retrieved from the UniProt. Homology models of the protein sequences were constructed using the SwissModel database for all 412 proteins. These were then refined through a protein blast and Ramachandran plot analysis. Out of the 412 constructed models, 143 models were of reliable quality. These were then submitted to the PMDB database for further reference. To demonstrate the application of these constructed models, protein-ligand docking analysis using Auto Dock Vina was performed. Among the antibiotics that were tested against their known drug targets, trimethoprim demonstrated significant potential for the inhibition of R. salmoninarum’s dihydrofolate reductase protein, with a binding energy of -9.06 Kcal/mol and with the formation of 3 hydrogen bonds. Therefore through protein-ligand docking studies and the construction of 3D models of protein drug targets, Trimethoprim is proposed as a solution to the Bacterial Kidney Disease (BKD) problem in salmonid fishes. Further in-vitro evidences are in demand to prove this hypothesis.