Interaction of CTX-M-15 enzyme with cefotaxime: a molecular modelling and docking study

Extended-spectrum β-lactamases (ESBLs) are the bacterial enzymes that make them resistant to advanced-generation cephalosporins. CTXM enzymes (the most prevalent ESBL-type) target cefotaxime. Aims of the study were: Modelling of CTX-M enzyme from bla(CTX-M) sequences of clinical Escherichia coli isolatesDocking of cefotaxime with modelled CTX-M enzymes to identify amino acid residues crucial to their interaction To hypothesize a possible relationship between 'interaction energy of the docked enzyme-antibiotic complex' and 'minimum inhibitory concentration (MIC) of the antibiotic against the bacteria producing that enzyme'. Seven E. coli strains of clinical origin which were confirmed as PCR-positive for bla(CTX-M) were selected for the study. C600 cells harboring cloned bla(CTX-M) were tested for ESBL-production by double-disk-synergy test. BLAST analysis confirmed all the bla(CTX-M) genes as blaCTX-M-15. Four of the 7 strains were found to be clonally related. Modelling was performed using Swiss Model Server. Discovery Studio 2.0 (Accelrys) was used to prepare Ramachandran plots for the modelled structures. Ramachandran Z-scores for modelled CTX-M enzymes from E. coli strains D8, D183, D253, D281, D282, D295 and D296 were found to be -0.449, 0.096, 0.027, 0.043, 0.032, -1.249 and -1.107, respectively. Docking was performed using Hex 5.1 and the results were further confirmed by Autodock 4.0. The amino acid residues Asn 104, Asn132, Gly 227, Thr 235, Gly 236, and Ser237 were found to be responsible for positioning cefotaxime into the active site of the CTX-M-15 enzyme. It was found that cefotaxime MICs for the CTX-M-15-producers increased with the increasing negative interaction energy of the enzyme-antibiotic complex.     

Identification of potential inhibitors for Klebsiella pneumoniae carbapenemase-3: a molecular docking and dynamics study

Abstract

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium, which is a leading causal agent for nosocomial infections. Penicillin, cephalosporin and carbapenems along with the inhibitors such as tazobactam, sulbactam and clavulanic acid are prescribed for the treatment of K. pneumoniae infections. Prolonged exposure to β-lactam antibiotics leads to the development of resistance. The major reason for the β-lactam resistance in K. pneumoniae is the secretion of the enzyme K. pneumoniae carbapenemase (KPC). Secretion of KPC-2 and its variant KPC-3 by the K. pneumoniae strains causes resistance to both the substrate imipenem and the β-lactamase inhibitors. Hence, molecular docking and dynamics studies were carried out to analyze the resistance mechanism of KPC-2–imipenem and KPC-3–imipenem at the structural level. It reveals that KPC-3-imipenem has the highest c-score value of 4.03 with greater stability than the KPC-2–imipenem c-score value of 2.36. Greater the interaction between the substrate and the β-lactamase enzyme, higher the chances of hydrolysis of the substrate. Presently available β-lactamase inhibitors are also ineffective against KPC-3-expressing strains. This situation necessitates the need for development of novel and effective inhibitors for KPC-3. We have carried out the virtual screening process to identify more effective inhibitors for KPC-3, and this has resulted in ZINC48682523, ZINC50209041 and ZINC50420049 as the best binding energy compounds, having greater binding affinity and stability than KPC-3–tazobactam interactions. Our study provides a clear understanding of the mechanism of drug resistance and provides valuable inputs for the development of inhibitors against KPC-3 expressing K. pneumoniae.

Communicated by Ramaswamy H. Sarma     

The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.     

Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: dihydrofolate monooxygenase activity

Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the K_M for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme.     

一株多重耐药鳗源肺炎克雷伯菌的分离鉴定

从患病花鳗鲡(Anguilla marmorata)的肝脏分离纯化到一株革兰氏阴性杆菌HM2。人工感染试验结果显示, HM2具有较强的致病力, 96h的LD50为9.98107 CFU/mL。经细菌培养特性、生理生化特性和ATB Expression半自动细菌鉴定仪鉴定, 结果符合肺炎克雷伯菌(Klebsiella pneumoniae)的特征。以细菌16S rRNA基因通用引物进行PCR扩增, 获得大小为1393 bp的部分16S rRNA基因序列(Genbank登录号为JX282908), 将所测序列与GenBank中的序列进行BLAST比对并构建系统进化树, 结果表明其与肺炎克雷伯菌的同源性最高(100%), 在系统发育树上与肺炎克雷伯菌聚为一簇, 进一步确定菌株HM2为肺炎克雷伯菌。药物敏感性试验显示, HM2对亚胺培南、链霉素、阿米卡星3种药物敏感, 对氨苄西林、阿莫西林/克拉维酸、头孢噻吩、头孢曲松、头孢噻肟、头孢西丁、复合磺胺、磺胺甲基异恶唑/甲氧苄啶、利福平、萘啶酸、环丙沙星、诺氟沙星、氧氟沙星、呋喃妥因、四环素、多西环素、氯霉素17种药物具有耐药性。研究结果为指导临床合理用药提供了科学依据。       

Cloning and characterization of dihydrofolate reductase from a facultative alkaliphilic and halotolerant bacillus strain

Elucidation of the molecular basis of the stability of enzymes from extremophilic organisms is of fundamental importance for various industrial applications. Due to the wealth of structural data from various species, dihydrofolate reductase (DHFR, EC 1.5.1.3) provides an excellent model for systematic investigations. In this report, DHFR from alkaliphilic Bacillus halodurans C-125 was cloned and expressed in E. coli. Functional analyses revealed that BhDHFR exhibits the most alkali-stable phenotype of DHFRs characterized so far. Optimal enzyme activity was observed in a slightly basic pH region ranging from 7.25 to 8.75. Alkali-stability is associated with a remarkable resistance to elevated temperatures (half-life of 60 min at 52.5°C) and to high concentrations of urea (up to 3 M). Although the secondary structure shows distinct similarities to those of mesophilic DHFR molecules, BhDHFR exhibits molecular features contributing to its alkaliphilic properties. Interestingly, the unique phenotype is diminished by C-terminal addition of a His-tag sequence. Therefore, His-tag-derivatized BhDHFR offers the opportunity to obtain deeper insights into the specific mechanisms of alkaliphilic adaption by comparison of the three dimensional structure of both BhDHFR molecules.     

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.     

Erratum to: Production of isobutanol from crude glycerol by a genetically-engineered Klebsiella pneumoniae strain

Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ?ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced isobutanol (160 mg l^?1) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l^?1. This represents the first successful attempt to produce isobutanol from crude glycerol.     

Interaction of procarbazine with calf thymus DNA—a biophysical and molecular docking study

Interaction of procarbazine (PCZ) with calf thymus DNA was studied using biophysical and molecular docking studies. Procarbazine was to interact with DNA with a binding constant of 6.52 × 10³ M⁻¹ as calculated using ultraviolet‐visible spectroscopy. To find out the binding mode, molecular docking was performed that predicted PCZ to interact with DNA through groove binding mode with binding affinity of −6.7 kcal/mole. To confirm the groove binding nature, different experiments were performed. Dye displacement assays confirmed the non‐intercalative binding mode. Procarbazine displaced Hoechst dye from the minor groove of DNA while it was unable to displace intercalating dyes. There was no increase in the viscosity of DNA solution in presence of PCZ. Also, negligible change in the secondary structure of DNA was observed in presence of PCZ as evident by circular dichroism spectra. Procarbazine caused decrease in the melting temperature of DNA possibly because of decrease in the stability of DNA caused by groove binding interaction of PCZ with DNA.     

Entry of methotrexate into Streptococcus pneumoniae: a study on a wild-type strain and a methotrexate resistant mutant

Entry of methotrexate (MTX) into the folate prototrophic bacterium Streptococcus pneumoniae was poorly inhibited by folate or its natural derivative folinic acid, suggesting that if MTX is transported via a folate transporter, the affinity of that transporter for MTX is higher than for folate. In the range of concentrations tested, MTX uptake was non-concentrative and decreased in ATP-depleted bacteria. When the external concentration of MTX was increased from 1 X 10(-7) M to 1 X 10(-6) M, uptake became saturated and was insensitive to ionophores. However when external MTX concentrations were increased to 1 X 10(-5) M, uptake increased linearly, and was inhibited by the ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, suggesting that the process was energized by the protonmotive force (delta p) at this concentration. A model for MTX entry in S. pneumoniae is proposed with respect to these results. The high level of resistance to MTX of the nonsense mutant amiA9 cannot be entirely explained by a decrease in MTX uptake.     

Electron redistribution on binding of a substrate to an enzyme: folate and dihydrofolate reductase

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum-mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to "electronic strain energy" in the substrate and enhanced protein-ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.     

Construction of a new catabolic pathway for d-fructose in Escherichia coli K12 using an l-sorbose-specific enzyme from Klebsiella pneumoniae

Starting with a fruK (formerly fpk) mutant of Escherichia coli K12 lacking d-fructose-1-phosphate kinase (E.C. 2.7.1.3.), fructose positive derivatives were isolated after introduction of the cloned gene sorE from Klebsiella pneumoniae coding for an l-sorbose-1-phosphate reductase. The new pathway was shwon to proceed from d-fructose via d-fructose-1-phosphate and d-mannitol-1-phosphate to d-fructose 6-phosphate. It involves a transport system and enzymes encoded in the fru and the mtl operons from E. coli K12 as well as in the sor operon from K. pneumoniae respectively.     

Diversity of aminoglycoside modifying enzyme genes among multidrug resistant Acinetobacter baumannii genotypes isolated from nosocomial infections in Tehran hospitals and their association with class 1 integrons

The aim of the present study was to investigate, for the first time, the diversity of the genes encoding aminoglycoside-modifying enzymes (AME) and their association with class 1 integrons in Iranian Acinetobacter baumannii strains. A total of 100 multidrug resistant A. baumannii, isolated from eight distinct hospitals in Tehran, were enrolled in this study. Susceptibility of these isolates to antimicrobial agents including gentamicin and amikacin was determined by E-test. Aminoglycoside resistant isolates were then tested by PCR for AME genes, including aphA6, aacC1, aacC2, aacA4, aadB, aadA1, classes 1 integron, 5'-CS-3' and typed by RAPD PCR. The rate of resistance to imipenem, meropenem, gentamicin and amikacin were 39%, 39%, 38% and 32%, respectively. Intermediate resistance phenotype to gentamicin and amikacin was observed in 2% and 5% of all the isolates, respectively. After aph6 with 90% (n = 36/40), aadA1, aacC1 and aadB with 82.5% (n = 33/40), 65% (n = 26/40) and 20% (n = 8/40) were the most prevalent AME genes among aminoglycosides resistant A. baumannii isolates. A combination of two to four different resistance genes was observed in 39 of 40 strains (97.5%), with a total of 7 different combinations. PCR of integrase genes revealed that AME gene was associated with 67% of class 1 integrons. RAPD analysis showed three predominant genotypes A (n = 20), B (n = 10) and 10 unrelated genotypes. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.     

Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.     

Study of reactivity of cyanoacetohydrazonoethyl-N-ethyl-N-methyl benzenesulfonamide: preparation of novel anticancer and antimicrobial active heterocyclic benzenesulfonamide derivatives and their molecular docking against dihydrofolate reductase

Abstract

This article describes the synthesis of some novel heterocyclic sulfonamides having biologically active thiophene 3, 4, 5, 6, coumarin 8, benzocoumarin 9, thiazole 7, piperidine 10, pyrrolidine 11, pyrazole 14 and pyridine 12, 13. Starting with 4-(1-(2-(2-cyanoacetyl)hydrazono)ethyl)-N-ethyl-N-methylbenzenesulfonamide (2), which was prepared from condensation of acetophenone derivative 1 with 2-cyanoacetohydrazide. The structures of the newly synthesized compounds were confirmed by elemental analysis, IR, ¹H NMR, ¹³C NMR, ¹⁹F NMR and MS spectral data. All the newly synthesized heterocyclic sulfonamides were evaluated as in-vitro anti-breast cancer cell line (MCF7) and as in-vitro antimicrobial agents. Compounds 8, 5 and 11 were more active than MTX reference drug and compounds 12, 7, 4, 14, 5 and 8 were highly potent against Klebsiella pneumonia. Molecular operating environment performed virtual screening using molecular docking studies of the synthesized compounds. The results indicated that some prepared compounds are suitable inhibitor against dihydrofolate reductase (DHFR) enzyme (PDBSD:4DFR) with further modification.     

Insight into the binding interactions of CYP450 aromatase inhibitors with their target enzyme: a combined molecular docking and molecular dynamics study

CYP450 aromatase catalyzes the terminal and rate-determining step in estrogen synthesis, the aromatization of androgens, and its inhibition is an efficient approach to treating estrogen-dependent breast cancer. Insight into the molecular basis of the interaction at the catalytic site between CYP450 aromatase inhibitors and the enzyme itself is required in order to design new and more active compounds. Hence, a combined molecular docking–molecular dynamics study was carried out to obtain the structure of the lowest energy association complexes of aromatase with some third-generation aromatase inhibitors (AIs) and with other novel synthesized letrozole-derived compounds which showed high in vitro activity. The results obtained clearly demonstrate the role of the pharmacophore groups present in the azaheterocyclic inhibitors (NSAIs)—namely the triazolic ring and highly functionalized aromatic moieties carrying H-bond donor or acceptor groups. In particular, it was pointed out that all of them can contribute to inhibition activity by interacting with residues of the catalytic cleft, but the amino acids involved are different for each compound, even if they belong to the same class. Furthermore, the azaheterocyclic group strongly coordinates with the Fe(II) of heme cysteinate in the most active NSAI complexes, while it prefers to adopt another orientation in less active ones.     

Molecular modelling of Mycobacterium tuberculosis acetolactate synthase catalytic subunit and its molecular docking study with inhibitors

Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, acetolactate synthase (mtALS) was selected as a target enzyme to combat M. tuberculosis. In this work, the three-dimensional molecular model of the hypothetical structure for the ALS catalytic subunit of M. tuberculosis was elucidated by homology modelling. In addition, the orientations and binding affinities of sulfonylurea inhibitors with the new structure was investigated. Our findings could be helpful for the design of new, more potent mtAHAS inhibitors.     

Interaction and photo-induced cleavage studies of a copper based chemotherapeutic drug with human serum albumin: spectroscopic and molecular docking study

The interaction of new dinuclear copper(ii) complex 1; [Cu(2)(glygly)(2)(ppz)(H(2)O)(4)]·2H(2)O, derived from dipeptide (glycyl glycine) and piperazine as a metallopeptide drug with human serum albumin (HSA) was examined by means of fluorescence spectroscopy which revealed that complex 1 has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The alterations of HSA secondary structure in the presence of complex 1 were confirmed by UV-visible, FT-IR, CD and 3D fluorescence spectroscopy. The binding constants (K), and binding site number (n), corresponding thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were calculated. The molecular docking technique was utilized to ascertain the mechanism and mode of action towards the molecular target HSA indicating that complex 1 was located at the entrance of site I by electrostatic and hydrophobic forces, consistent with the corresponding experimental results. Complex 1 shows efficient photo-induced HSA cleavage activity, indicating the involvement of hydroxyl radicals as the reactive species. Furthermore, the cytotoxicity of 1 was examined on a panel of human tumor cell lines of different histological origins showing significant GI(50) values specifically towards MIAPACA2, A498 and A549 tumor cell lines. These results complement previous biological studies of new specific target metallopeptides, providing additional information about possibilities of their transport and disposition in blood plasma.