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The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for an immediate search for novel treatment strategies. Recently, BlaC, the principal beta-lactamase of Mycobacterium tuberculosis , was recognized as a potential therapeutic target. BlaC belongs to Ambler class A, which is generally susceptible to the beta-lactamase inhibitors currently used in clinics: tazobactam, sulbactam, and clavulanate. Alterations at Ser130 in conserved SDN loop confer resistance to mechanism-based inhibitors (MBIs) commonly observed in various clinical isolates. The absence of clinical evidence of S130G conversion in M. tuberculosis draws our attention to build laboratory mutants of S130G and S130A of BlaC. The study involving steady state, inhibition kinetics, and fluorescence microscopy shows the emergence of resistance against MBIs to the mutants expressing S130G and S130A. To understand the molecular reasoning behind the unavailability of such mutation in real life, we have used circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), molecular dynamics (MD) simulation, and stability-based enzyme activity to compare the stability and dynamic behaviors of native and S130G/A mutant form of BlaC. A significant decrease in melting temperature (BlaC T M 60°C, S130A T M 50°C, and S130G T M 45°C), kinetic instability at higher temperature, and comparative dynamic instability correlate the fact that resistance to beta-lactam/beta-lactamase inhibitor combinations will likely not arise from the structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be potentially applied as a part of a successful treatment regimen against M. tuberculosis. [ABSTRACT FROM AUTHOR]

Vitreous or vitreous humor is a complex transparent gel that fills the space between the lens and retina of an eye and acts as a transparent medium that allows light to pass through it to reach the photoreceptor layer (retina) of the eye. The vitreous humor is removed in ocular surgery (vitrectomy) for pathologies like retinal detachment, macular hole, diabetes‐related vitreous hemorrhage detachment, and ocular trauma. Since the vitreous is not actively regenerated or replenished, there is a need for a vitreous substitute to fill the vitreous cavity to provide a temporary or permanent tamponade to the retina following some vitreoretinal surgeries. An ideal vitreous substitute could probably be left inside the eye forever. The vitreous humor is transparent, biocompatible, viscoelastic and highly hydrophilic; polymeric hydrogels with these properties can be a potential candidate to be used as vitreous substitutes. To meet the tremendous demand for the vitreous substitute, many scientists all over the world have developed various kinds of vitreous substitutes or tamponade agent. Vitreous substitutes, whatsoever developed till date, are associated with several advantages and disadvantages, and there is no ideal vitreous substitute available till date. This review highlights the polymer‐based vitreous substitutes developed so far, along with their advantages and limitations. The gas‐based and oil‐based substitutes have also been discussed but very briefly. [ABSTRACT FROM AUTHOR]

A new monomeric copper(II) benzhydrazone complex, [Cu(L)(H2O)(NO3)] (1), has been synthesized using Schiff base derived from quinoline-2-carboxaldehyde and benzhydrazide. Complex 1 was characterized by elemental analysis, FT-IR, UV-Vis spectroscopy along with electrochemical study and single-crystal X-ray crystallography. Single-crystal diffraction study of 1 indicates that the copper(II) ion adopts a distorted square pyramidal (SQP) geometry. The calf-thymus (CT) DNA-binding property of 1 has been evaluated by employing UV-vis and fluorescence spectroscopy, cyclic voltammetry and viscosity studies. The protein-binding ability of 1 was investigated by UV-vis and fluorescence spectroscopy titration using BSA and HSA. The significant interaction of 1 with CT-DNA was further supported by molecular docking approach. Finally, in vitro cytotoxicity of 1 was investigated by MTT assay on the Human Skin Carcinoma cell line A431. All these facts indicate that 1 exhibits a promising anticancer activity. [ABSTRACT FROM AUTHOR]

The crystal structure of human deoxycytidine kinase (dCK) in complex with the cytidine analogue 5-methyldeoxycytidine (5-Me-dC) is solved in order to understand the determinants preventing thymidine phosphorylation by wild-type (WT) dCK. The studies have shown that it is in fact residue Asp133 of dCK that is most responsible for discriminating against the thymine base and dCK variants have improved activity with 5-substituted deoxycytidine and thymidine analogoues.

The kinetic analysis of human deoxycytidine kinase (dCK) variants is performed to mutate and select the optimal residues at position 104 and an alanine at position 133. The unique catalytic properties observed for several of the mutants suggested them to be good candidates for suicide-gene/protein-therapy applications.

Epithelial–mesenchymal transition (EMT) is a key step in transdifferentiation process in solid cancer development. Forthcoming evidence suggest that the stratified program transforms polarized, immotile epithelial cells to migratory mesenchymal cells associated with enhancement of breast cancer stemness, metastasis, and drug resistance. It involves primarily several signaling pathways, such as transforming growth factor‐β (TGF‐β), cadherin, notch, plasminogen activator protein inhibitor, urokinase plasminogen activator, and WNT/beta catenin pathways. However, current understanding on the crosstalk of multisignaling pathways and assemblies of key transcription factors remain to be explored. In this review, we focus on the crosstalk of signal transduction pathways linked to the current therapeutic and drug development strategies. We have also performed the computational modeling on indepth the structure and conformational dynamic studies of regulatory proteins and analyze molecular interactions with their associate factors to understand the complicated process of EMT in breast cancer progression and metastasis. Electrostatic potential surfaces have been analyzed that help in optimization of electrostatic interactions between the protein and its ligand. Therefore, understanding the biological implications underlying the EMT process through molecular biology with biocomputation and structural biology approaches will enable the development of new therapeutic strategies to sensitize tumors to conventional therapy and suppress their metastatic phenotype. [ABSTRACT FROM AUTHOR]

Breast cancer is found to be the most prevalent neoplasm in women worldwide. Despite the function of physically tethering cells to the matrix, transmembrane protein integrins are crucially involved in diverse cellular functions such as cell differentiation, proliferation, invasion, migration, and metastasis. Dysregulation of integrins and their interactions with the cells and their microenvironment can trigger several signaling cues that determine the cell fate decision. In this review, we spotlight all pre-existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumors and their microenvironment to conventional therapy and overall suppress their metastatic phenotype. [ABSTRACT FROM AUTHOR]

Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart. [ABSTRACT FROM AUTHOR]

The genome of Mycobacterium tuberculosis contains a gene, blaC, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme-drug covaient complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent Km and Kcat values of 0.8 μM and 0.03 min-1, respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl-enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC-tebipenem covaient acylated adduct (1.9 Å) with wild-type BlaC and the BlaC-tebipenem Michaelis- Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem- BlaC structures. [ABSTRACT FROM AUTHOR]

The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occur in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, N -acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively nonlabile ether bond and presents an interesting target for mechanistic studies. Open chain product and substrate analogues were synthesized and tested as competitive inhibitors (Kis is values of 1.1 ± 0.3 and 0.23 ± 0.02 mM, respectively) of the MurNAc 6P hydrolase from Escherichia coli (MurQ-EC). To identify the roles of active site residues that are important for catalysis, the substrate analogue was cocrystallized with the MurNAc 6P hydrolase from Haemophilus influenzae (MurQ-HI) that was amenable to crystallographic studies. The cocrystal structure of MurQ-HI with the substrate analogue showed that Glu89 was located in the proximity of both the C2 atom and the oxygen at the C3 position of the bound inhibitor and that no other potential acid/ base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring opening. Combining these results with previous biochemical data, we propose a one-base mechanism of action in which Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [D2OV = 3.0 ± 0.2, and D2OV/Kisocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [DV = 1.3 ± 0.1, and D(V/K[2R-²H]isocitrate) = 15 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn2+ bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking β sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of αa-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel. [ABSTRACT FROM AUTHOR]

NXL104 is a novel β-lactamase inhibitor with a non-lactam structural scaffold. Our kinetic and mass spectrometric analysis demonstrates that NXL104 quantitatively inhibits BlaC, the only chromosomally encoded β-lactamase from Mycobacterium tuberculosis, by forming a carbamyl adduct with the enzyme. The inhibition efficiency (k2/K) of NXL104 was shown to be more than 100-fold lower than that of clavulanate, a classical β-lactamase inhibitor, which is probably caused by the bulky rings of NXL104. However, the decarbamylation rate constant (k3) was determined to be close to zero. The BlaC—NXL104 adduct remained stable for at least 48 h, while the hydrolysis of the BlaC—clavulanate adduct was observed after 2 days. The three-dimensional crystal structure of the BlaC—NXL104 carbamyl adduct was determined at a resolution of 2.3 Å. Interestingly, the sulfate group of NXL104 occupies the position of a phosphate ion in the structure of the BlaC—clavulanate adduct and is hydrogen bonded to residues Ser128, Thr237, and Thr239. Favorable interactions are also seen in the electrostatic potential map. We propose that these additional interactions, as well as the intrinsic stability of the carbamyl linkage, contribute to the extraordinary stability of the BlaC—NXL104 adduct. [ABSTRACT FROM AUTHOR]

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant. [ABSTRACT FROM AUTHOR]

Abstract: Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k cat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. [Copyright &y& Elsevier]