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Naidu Subbarao - One of the best experts on this subject based on the ideXlab platform.
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Design and in vitro analysis of SIRT2 inhibitor targeting Parkinson’s disease
Molecular Diversity, 2020Co-Authors: Amrendra Pratap Singh, Lokesh Nigam, Yudhishthir Yadav, Shashank Shekhar, Naidu SubbaraoAbstract:Inhibition of Sirtuin2 (SIRT2) protein rescues the α -synuclein toxicity in vitro and in vivo models of Parkinson’s disease (PD). Thioacetyl group can structurally mimic the acetyl group and restrain the deacetylating p53 reaction by SIRT2. This work evaluated the biological activity of designed Pentapeptides inhibitor containing N -thioacetyl-lysine against SIRT2. Pentapeptide by introducing thioacetyl-lysine as an inhibitor of SIRT2 was screened by molecular docking and synthesized by solid phase method. The inhibition of pure recombinant SIRT2 as well as SIRT2 in serum of PD patients by peptide was done by fluorescent activity assay. The inhibition of SIRT2 was assessed in PC12 cell line by measuring acetylated α -tubulin level. The peptide YKK( ε -thioAc)AM and HRK( ε -thioAc)AM were found to be SIRT2 inhibitors by molecular docking. However, YKK( ε -thioAc)AM was more specific towards SIRT2 than SIRT1 (Sirtuin1). It inhibited recombinant SIRT2 by IC_50 value of 0.15 µM and KD values 9.92 × 10^−8/M. It also inhibited serum SIRT2 of PD. It increased the acetylation of α -tubulin in PC12 neuroblastoma cells which is essential for maintaining the microtubular cell functions of brain. It can be concluded that novel peptide YKK( ε -thioAc)AM may be a platform for therapeutic agent for Parkinson’s disease targeting SIRT2. Graphic abstract
Hirokazu Tamamura - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship studies on cxcr4 antagonists having cyclic Pentapeptide scaffolds
Organic and Biomolecular Chemistry, 2005Co-Authors: Hirokazu Tamamura, Ai Esaka, Teppei Ogawa, Takanobu Araki, Satoshi Ueda, Zixuan Wang, John O Trent, Hiroshi Tsutsumi, Hiroyuki MasunoAbstract:Structure–activity relationship studies on CXCR4 antagonists, which were previously found by using cyclic Pentapeptide libraries, were performed to optimize side-chain functional groups, involving conformationally constrained analogues. In addition, a new lead of cyclic Pentapeptides with the introduction of a novel pharmacophore was developed.
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Structure–activity relationship studies on CXCR4 antagonists having cyclic Pentapeptide scaffolds
Organic and Biomolecular Chemistry, 2005Co-Authors: Hirokazu Tamamura, Ai Esaka, Teppei Ogawa, Takanobu Araki, Satoshi Ueda, Zixuan Wang, John O Trent, Hiroshi Tsutsumi, Hiroyuki Masuno, Hideki NakashimaAbstract:Structure–activity relationship studies on CXCR4 antagonists, which were previously found by using cyclic Pentapeptide libraries, were performed to optimize side-chain functional groups, involving conformationally constrained analogues. In addition, a new lead of cyclic Pentapeptides with the introduction of a novel pharmacophore was developed.
Brian L Mark - One of the best experts on this subject based on the ideXlab platform.
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the β lactamase gene regulator ampr is a tetramer that recognizes and binds the d ala d ala motif of its repressor udp n acetylmuramic acid murnac Pentapeptide
Journal of Biological Chemistry, 2015Co-Authors: Grishma Vadlamani, David J. Vocadlo, Misty D Thomas, Trushar R Patel, Lynda J Donald, Thomas M Reeve, Jorg Stetefeld, Kenneth G Standing, Brian L MarkAbstract:Inducible expression of chromosomal AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to β-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-Pentapeptide. When exposed to β-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-Pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-Pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-Pentapeptide revealed that the terminal d-Ala-d-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-Pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the d-Ala-d-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-Pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-Pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function.
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inactivation of the glycoside hydrolase nagz attenuates antipseudomonal β lactam resistance in pseudomonas aeruginosa
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Azizah Asgarali, Keith A. Stubbs, Antonio Oliver, David J. Vocadlo, Brian L MarkAbstract:Pseudomonas aeruginosa is a versatile gram-negative bacterium that is ubiquitous in the environment. Over the last century, it has emerged as one of the most significant opportunistic pathogens of humans and now accounts for over 10% of all hospital-acquired infections (10, 35, 41). P. aeruginosa exhibits high levels of intrinsic resistance to antibiotics, and P. aeruginosa infections are often persistent and associated with considerable morbidity and mortality (12, 36). P. aeruginosa is a leading cause of nosocomial pneumonia, urinary tract infections, and secondary bacteremia associated with burn wounds (36, 46). Moreover, environmental reservoirs of P. aeruginosa play a primary role in the morbidity and mortality of patients with cystic fibrosis (CF) by chronically colonizing the lungs of these patients (35). Nearly 80% of patients with CF become infected with this microbe by early adulthood (9, 13, 23). Many antibiotics initially overcome the intrinsic drug resistance mechanisms of P. aeruginosa; however, all clinically relevant therapies can be compromised by the generation of drug-resistant genetic mutants (28). Intrinsic resistance to β-lactam antibiotics occurs via the induction of chromosomally encoded AmpC β-lactamase (19, 29). The degree of resistance to β-lactams depends on the level of ampC gene induction; although they are susceptible to hydrolysis by AmpC, some penicillins (such as piperacillin) and cephalosporins (such as cefepime or ceftazidime) exhibit antipseudomonal activity because they are weak inducers of ampC expression (27). However, the prolonged use of antipseudomonal β-lactams frequently selects for mutants that hyperproduce AmpC β-lactamase, which often leads to the failure of treatment with these antibiotics (11, 28). Inducible chromosomal ampC has been identified in a number of enterobacteria and in P. aeruginosa. The regulation of ampC induction in these microorganisms is closely coupled to cell wall peptidoglycan (PG) recycling (Fig. (Fig.1)1) (27, 32, 33). During growth, periplasmic autolysins process a considerable amount of PG into GlcNAc-1,6-anhydromuropeptide (tri-, tetra-, and Pentapeptide) fragments (for a review, see reference 49). These fragments are transported into the cytosol (4, 6), where the nonreducing GlcNAc residue is removed by a family 3 (14) glycoside hydrolase encoded by nagZ (3, 50). The resulting products are GlcNAc and a pool of cytosolic 1,6-anhydro-MurNAc peptides (tri-, tetra-, and Pentapeptides) (3, 50), which are normally recycled into UDP-MurNAc Pentapeptide, a PG precursor that is exported to the periplasm and reincorporated back into the cell wall. FIG. 1. Schematic of the PG recycling pathway and its role in AmpC β-lactamase induction. During growth, GlcNAc-1,6-anhydro-MurNAc tri-, tetra-, and Pentapeptides (only the tripeptide species is shown) are excised from the PG and transported into the ... From the pool of 1,6-anhydro-MurNAc peptide catabolites, either the tripeptide species (17) or the Pentapeptide species (7) is believed to be the signaling molecule that induces ampC transcription, whereas the anabolic product UDP-MurNAc Pentapeptide acts to repress ampC transcription (Fig. (Fig.1).1). These metabolites are thought to competitively regulate ampC transcription by directly binding to a LysR-type transcriptional regulator encoded by ampR (17). Together, ampR and ampC form a divergent operon with overlapping promoter regions to which AmpR binds and thereby regulates the transcription of both genes (2, 26). The relative levels of these metabolites govern whether ampC is transcribed. Under normal growth conditions, the cytosolic concentration of 1,6-anhydro-MurNAc peptide is suppressed by the activity of AmpD, a cytoplasmic N-acetyl-muramyl-l-alanine amidase that cleaves the stem peptide off from both GlcNAc-1,6-anhydro-MurNAc peptide and 1,6-anhydro-MurNAc peptide (16, 18). The low cellular level of these inducer molecules therefore allows UDP-MurNAc Pentapeptide to bind to AmpR and promote the formation of an AmpR-DNA complex that represses ampC transcription. Exposure to β-lactams, however, elevates the level of PG fragmentation (6, 34, 48) to levels that cannot be efficiently processed by endogenous AmpD activities, allowing the NagZ products 1,6-anhydro-MurNAc tripeptide (or Pentapeptide) to accumulate and presumably competitively displace UDP-MurNAc Pentapeptide from AmpR, generating a complex that acts as a transcriptional activator of ampC (17). Although antipseudomonal β-lactams, such as ceftazidime, piperacillin, and cefepime, are not susceptible to this intrinsic resistance mechanism, the selection of loss-of-function mutations in ampD (20, 22, 25, 40) shunts PG recycling toward the accumulation of cytosolic 1,6-anhydro-MurNAc peptide and causes the derepression of ampC at a level of that is sufficient to confer resistance to even these β-lactams (16, 21). P. aeruginosa has recently been found to encode three ampD homologues: ampD, ampDh2, and ampDh3. All three appear to work in concert to repress ampC induction (21); the stepwise deletion of ampD, ampDh2, and ampDh3 results in a three-step upregulation mechanism of ampC expression, with the triple null mutant exhibiting complete derepression of chromosomal AmpC β-lactamase (21). The presence of multiple ampD homologues appears to provide P. aeruginosa the ability to acquire resistance to β-lactams through the partial derepression of ampC expression via loss-of-function mutations in ampD even while maintaining its fitness and virulence by sustaining PG recycling via the activities of AmpDh2 and AmpDh3 (31). Recently, a β-lactam-resistant P. aeruginosa isolate from the lung of a CF patient was found to contain loss-of-function mutations in both ampD and ampDh3 (37); however, the inactivation of multiple ampD homologues may be uncommon, since the constitutive hyperexpression of ampC has been linked to reduced fitness (30, 31). Given that NagZ catalyzes the formation of the inducer molecule 1,6-anhydro-MurNAc tripeptide (or Pentapeptide), inhibition of the activity of this enzyme in P. aeruginosa may provide an effective strategy to prophylactically repress ampC expression during β-lactam therapy or to enhance the efficacy of antipseudomonal penicillins and cephalosporins against resistant mutants containing ampD null mutations. We recently demonstrated that a series of selective small-molecules inhibitors targeting NagZ could repress ampC induction in an Escherichia coli model system harboring the ampC-ampR operon from Citrobacter freundii (42). This model system was previously used to demonstrate that the NagZ function is required for the production of AmpC β-lactamase from a plasmid-borne ampC-ampR operon (50). The importance and functional role, however, of NagZ in gram-negative pathogens with a chromosomally encoded ampC-ampR operon have not yet been investigated. To understand the role of NagZ in the AmpC β-lactamase induction pathway of P. aeruginosa, we have inactivated nagZ (PA3005) in P. aeruginosa reference strain PAO1 (41) and in an AmpD-deficient strain of PAO1 (strain PAΔDDh2Dh3, in which ampD [D], ampDh2 [Dh2], and ampDh3 [Dh3] are inactivated) (21) (Table (Table1).1). We have measured the sensitivities of the nagZ null mutants to antipseudomonal β-lactams and demonstrate that the inactivation of nagZ reduces both intrinsic β-lactam resistance and the high antipseudomonal β-lactam resistance associated with the loss of AmpD activity. We also demonstrate that AmpC-mediated resistance to antipseudomonal β-lactams can be suppressed in P. aeruginosa by using a potent and selective small-molecule inhibitor of NagZ. The results suggest that the blockage of NagZ activity could provide an effective strategy to enhance the efficacies of β-lactams against gram-negative pathogens encoding inducible chromosomal AmpC and counteract the hyperinduction of AmpC β-lactamase that occurs from the selection of ampD mutants during β-lactam therapy. TABLE 1. Plasmids and bacterial strains
J Van Heijenoort - One of the best experts on this subject based on the ideXlab platform.
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the murg gene of escherichia coli codes for the udp n acetylglucosamine n acetylmuramyl Pentapeptide pyrophosphoryl undecaprenol n acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis
Journal of Bacteriology, 1991Co-Authors: Dominique Menginlecreulx, L Texier, M Rousseau, J Van HeijenoortAbstract:Abstract Physiological properties of the murG gene product of Escherichia coli were investigated. The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-Pentapeptide (UDP-MurNAc-Pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-Pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors. The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphoryl-MurNAc-(Pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I. In vitro assays for phospho-MurNAc-Pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway. Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed. In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.
David P. Fairlie - One of the best experts on this subject based on the ideXlab platform.
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Comparative α‐Helicity of Cyclic Pentapeptides in Water
Angewandte Chemie, 2014Co-Authors: Aline Dantas De Araujo, Huy N. Hoang, Frederik Diness, Praveer Gupta, Timothy A. Hill, Russell W. Driver, David Price, Spiros Liras, David P. FairlieAbstract:Helix-constrained polypeptides have attracted great interest for modulating protein–protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1–X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model Pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 310-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic Pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide.
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single turn peptide alpha helices with exceptional stability in water
Journal of the American Chemical Society, 2005Co-Authors: Nicholas E Shepherd, Huy N. Hoang, Giovanni Abbenante, David P. FairlieAbstract:Cyclic Pentapeptides are not known to exist in α-helical conformations. CD and NMR spectra show that specific 20-membered cyclic Pentapeptides, Ac−(cyclo-1,5) [KxxxD]-NH2 and Ac-(cyclo-2,6)-R[KxxxD]-NH2, are highly α-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest α-helical peptides in water.
