This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM 2 ) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM 2 in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM 2 by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM 2 showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM 2 by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.A variety of genetic diseases and experimental situations within the heterozygous state depict an allelic relationship where the mutant recessive allele overrides the function of its normal (wild type) dominant allele. This condition, referred to as dominant negative, is usually observed in the case of oligomeric or multidomain proteins with a possibility of cross-monomer interaction. Such mutant proteins by acting as competitive inhibitors of the normal protein function could generate polymorphic forms in a single cell. A phenomenon observed in collagen, where dominant negative mutations cause the production of abnormal oligomers (1, 2), and in transcription factors like helix-loop-helix and leucine zippers, where mutant monomers sequestering the function of wild type in a dimer bound to DNA, leads to an altered gene expression (3-5). Dominant negative mutations are also reported to affect some multifunctional molecules like p53 with differential impact on cell physiology (6 -10).Pyruvate kinase (EC 2.7.1.40) catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP-generating pyruvate and ATP in glycolysis (11,12). Depending upon the differential metabolic requirements of the tissues, the enzyme is expressed in four different isoforms, L, R, M 1 , and M 2 in vertebrates (13). PKM 2 2 is a ubiquitous, prototype enzyme, present in all tissues during embryonic stage, and is gradually replaced by other isozymic forms in specific tissues, during development. It is necessary for cellular division irrespective of the type of tissue and reappears during cellular division and tumor formation (14 -17). PKM 2 is known to regulate its activity by switching between an active tetramer and inactive dimer form in a fructose 1,6-bisphosphate-dependent manner to shift the cellular met...

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The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (K i = 0.017 µM) and better acylation efficiency (k +2/K′ = 0.44 µM−1s−1). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s−1). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.

In this population with a normal cervical cytology, the prevalence of high-risk HPV types was very low. The major prevalent HPV genotype in Punjab Province of Pakistan was the low-risk HPV type 6, followed by HPV type 55. Sequencing of the partial L1 region suggested that the region was highly conserved in all reported sequences. This study highlights the need to conduct robust epidemiological studies in the region and to develop regular HPV screening so that the situation does not reach an alarming stage resulting in cervical cancer.

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The global rise of metallo-β-lactamases (MBLs) is problematic due to their ability to inactivate most β-lactam antibiotics. MBL inhibitors that could be coadministered with and restore the efficacy of β-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes, we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from an approximately 1.5 million compound library, 6 exhibited IC values less than 40 μM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC values of 19 ± 2, 14 ± 1, and 50 ± 20 μM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B β-lactamase (BBL) numbering scheme is also included.

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