Co-reporter:Sebastián A. Testero, Carla Granados, Daniel Fernández, Pablo Gallego, Giovanni Covaleda, David Reverter, Josep Vendrell, Francesc X. Avilés, Irantzu Pallarès, and Shahriar Mobashery
ACS Medicinal Chemistry Letters October 12, 2017 Volume 8(Issue 10) pp:1122-1122
Publication Date(Web):September 22, 2017
DOI:10.1021/acsmedchemlett.7b00346
Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer’s disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.Keywords: Carboxypeptidase A; mechanism-based inactivators; thiiranes; X-ray crystallography;
Co-reporter:Kiran V. Mahasenan, Maria Bastian, Ming Gao, Emma Frost, Derong Ding, Katerina Zorina-Lichtenwalter, John Jacobs, Mark A. Suckow, Valerie A. Schroeder, William R. Wolter, Mayland Chang, and Shahriar Mobashery
ACS Medicinal Chemistry Letters June 8, 2017 Volume 8(Issue 6) pp:654-654
Publication Date(Web):May 1, 2017
DOI:10.1021/acsmedchemlett.7b00130
Matrix metalloproteinases (MMPs) have numerous physiological functions and share a highly similar catalytic domain. Differential dynamical information on the closely related human MMP-8, -13, and -14 was integrated onto the benzoxazinone molecular template. An in silico library of 28,099 benzoxazinones was generated and evaluated in the context of the molecular-dynamics information. This led to experimental evaluation of 19 synthesized compounds and identification of selective inhibitors, which have potential utility in delineating the physiological functions of MMPs. Moreover, the approach serves as an example of how dynamics of closely related active sites may be exploited to achieve selective inhibition by small molecules and should find applications in other enzyme families with similar active sites.Keywords: Animal studies; Enzyme kinetics; Matrix metalloproteinases; Molecular docking; Molecular dynamics; Virtual library design;
Co-reporter:Teresa Domínguez-Gil, Rafael Molina, David A. Dik, Edward Spink, Shahriar Mobashery, and Juan A. Hermoso
Biochemistry December 5, 2017 Volume 56(Issue 48) pp:6317-6317
Publication Date(Web):November 13, 2017
DOI:10.1021/acs.biochem.7b00932
Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1.
Co-reporter:David A. Dik, Teresa Domínguez-Gil, Mijoon Lee, Dusan Hesek, Byungjin Byun, Jennifer FishovitzBill Boggess, Lance M. Hellman, Jed F. Fisher, Juan A. Hermoso, Shahriar Mobashery
Journal of the American Chemical Society 2017 Volume 139(Issue 4) pp:1448-1451
Publication Date(Web):January 12, 2017
DOI:10.1021/jacs.6b12819
A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 μM. The EBD binds to the suppressor ligand UDP-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a “closed” conformation, in contrast to the “open” structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.
Co-reporter:Kiran V. MahasenanRafael Molina, Renee Bouley, María T. Batuecas, Jed F. Fisher, Juan A. Hermoso, Mayland Chang, Shahriar Mobashery
Journal of the American Chemical Society 2017 Volume 139(Issue 5) pp:2102-2110
Publication Date(Web):January 18, 2017
DOI:10.1021/jacs.6b12565
The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a—each with the β-lactam in the allosteric site—defined (with preceding PBP2a structures) as the “open” or “partially open” PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known “closed” apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for β-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.
Co-reporter:Mijoon Lee;Dusan Hesek;Jaroslav Zajíček;Jed F. Fisher
Chemical Communications 2017 vol. 53(Issue 95) pp:12774-12777
Publication Date(Web):2017/11/28
DOI:10.1039/C7CC06781J
The repeating isoprene unit is a fundamental biosynthetic motif. The repetitive structure presents challenges both for synthesis and for structural characterization. In this synthesis of the (Z8,E2,ω)-undecaprenol of prokaryotic glycobiology, we exemplify solutions to these challenges. Allylation of sulfone-derived carbanions controlled the stereochemistry, and its proof-of-structure was secured by Eu(hfc)3 complexation to disperse the overlaid resonances of its 1H NMR spectrum.
Co-reporter:Dr. Mijoon Lee;Dr. Dusan Hesek;David A. Dik;Dr. Jennifer Fishovitz;Elena Lastochkin;Dr. Bill Boggess;Dr. Jed F. Fisher; Shahriar Mobashery
Angewandte Chemie 2017 Volume 129(Issue 10) pp:2779-2783
Publication Date(Web):2017/03/01
DOI:10.1002/ange.201611279
AbstractAn enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.
Co-reporter:Renee Bouley; Derong Ding; Zhihong Peng; Maria Bastian; Elena Lastochkin; Wei Song; Mark A. Suckow; Valerie A. Schroeder; William R. Wolter; Shahriar Mobashery;Mayland Chang
Journal of Medicinal Chemistry 2016 Volume 59(Issue 10) pp:5011-5021
Publication Date(Web):April 18, 2016
DOI:10.1021/acs.jmedchem.6b00372
We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity against Staphylococcus aureus. The first structure–activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.
Co-reporter:Mijoon Lee, Teresa Domínguez-Gil, Dusan Hesek, Kiran V. Mahasenan, Elena Lastochkin, Juan A. Hermoso, and Shahriar Mobashery
ACS Chemical Biology 2016 Volume 11(Issue 6) pp:1525
Publication Date(Web):April 1, 2016
DOI:10.1021/acschembio.6b00194
A family of 11 lytic transglycosylases in Pseudomonas aeruginosa, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair, and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of P. aeruginosa is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover.
Co-reporter:Huan Wang; Dusan Hesek; Mijoon Lee; Elena Lastochkin; Allen G. Oliver; Mayland Chang
Journal of Natural Products 2016 Volume 79(Issue 4) pp:1219-1222
Publication Date(Web):April 6, 2016
DOI:10.1021/acs.jnatprod.6b00057
Four possible isomers of essramycin, a natural product from a marine Streptomyces species isolated from the Egyptian Mediterranean coast, were synthesized. The structures for the isomers were assigned unequivocally by 1H NMR, 13C NMR, high-resolution mass spectrometry, and X-ray crystal structure determinations. Notwithstanding the earlier report of broad-spectrum antibacterial activity for the natural product, none of the four isomers exhibited any such activity.
Co-reporter:Erika Leemans, Kiran V. Mahasenan, Malika Kumarasiri, Edward Spink, Derong Ding, Peter I. O’Daniel, Marc A. Boudreau, Elena Lastochkin, Sebastian A. Testero, Takao Yamaguchi, Mijoon Lee, Dusan Hesek, Jed F. Fisher, Mayland Chang, Shahriar Mobashery
Bioorganic & Medicinal Chemistry Letters 2016 Volume 26(Issue 3) pp:1011-1015
Publication Date(Web):1 February 2016
DOI:10.1016/j.bmcl.2015.12.041
The oxadiazole antibacterials, a class of newly discovered compounds that are active against Gram-positive bacteria, target bacterial cell-wall biosynthesis by inhibition of a family of essential enzymes, the penicillin-binding proteins. Ligand-based 3D-QSAR analyses by comparative molecular field analysis (CoMFA), comparative molecular shape indices analysis (CoMSIA) and Field-Based 3D-QSAR evaluated a series of 102 members of this class. This series included inactive compounds as well as compounds that were moderately to strongly antibacterial against Staphylococcus aureus. Multiple models were constructed using different types of energy minimization and charge calculations. CoMFA derived contour maps successfully defined favored and disfavored regions of the molecules in terms of steric and electrostatic properties for substitution.
Co-reporter:Jennifer Fishovitz; Negin Taghizadeh; Jed F. Fisher; Mayland Chang
Journal of the American Chemical Society 2015 Volume 137(Issue 20) pp:6500-6505
Publication Date(Web):May 12, 2015
DOI:10.1021/jacs.5b01374
The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-d-Ala-d-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper–Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-d-Ala-d-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-d-Ala-d-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA.
Co-reporter:Renee Bouley; Malika Kumarasiri; Zhihong Peng; Lisandro H. Otero; Wei Song; Mark A. Suckow; Valerie A. Schroeder; William R. Wolter; Elena Lastochkin; Nuno T. Antunes; Hualiang Pi; Sergei Vakulenko; Juan A. Hermoso; Mayland Chang
Journal of the American Chemical Society 2015 Volume 137(Issue 5) pp:1738-1741
Publication Date(Web):January 28, 2015
DOI:10.1021/jacs.5b00056
In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.
Co-reporter:Edward Spink; Derong Ding; Zhihong Peng; Marc A. Boudreau; Erika Leemans; Elena Lastochkin; Wei Song; Katerina Lichtenwalter; Peter I. O’Daniel; Sebastian A. Testero; Hualiang Pi; Valerie A. Schroeder; William R. Wolter; Nuno T. Antunes; Mark A. Suckow; Sergei Vakulenko; Mayland Chang
Journal of Medicinal Chemistry 2015 Volume 58(Issue 3) pp:1380-1389
Publication Date(Web):January 15, 2015
DOI:10.1021/jm501661f
The structure–activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.
Co-reporter:Huan Wang; Mijoon Lee; Zhihong Peng; Blas Blázquez; Elena Lastochkin; Malika Kumarasiri; Renee Bouley; Mayland Chang
Journal of Medicinal Chemistry 2015 Volume 58(Issue 10) pp:4194-4203
Publication Date(Web):April 29, 2015
DOI:10.1021/jm501831g
Rapid emergence of antibiotic resistance is one of the most challenging global public health concerns. In particular, vancomycin-resistant Enterococcus faecium infections have been increasing in frequency, representing 25% of enterococci infections in intensive care units. A novel class of 1,2,4-triazolo[1,5-a]pyrimidines active against E. faecium is reported herein. We used a three-component Biginelli-like heterocyclization reaction for the synthesis of a series of these derivatives based on reactions of aldehydes, β-dicarbonyl compounds, and 3-alkylthio-5-amino-1,2,4-triazoles. The resulting compounds were assayed for antimicrobial activity against the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These analyses identified a subset of 1,2,4-triazolo[1,5-a]pyrimidines that had good narrow-spectrum antibacterial activity against E. faecium and exhibited metabolic stability with low intrinsic clearance. Macromolecular synthesis assays revealed cell-wall biosynthesis as the target of these antibiotics.
Co-reporter:Marc A. Boudreau, Jennifer Fishovitz, Leticia I. Llarrull, Qiaobin Xiao, and Shahriar Mobashery
ACS Infectious Diseases 2015 Volume 1(Issue 10) pp:454
Publication Date(Web):August 18, 2015
DOI:10.1021/acsinfecdis.5b00086
Methicillin-resistant Staphylococcus aureus (MRSA), an important human pathogen, has evolved an inducible mechanism for resistance to β-lactam antibiotics. We report herein that the integral membrane protein BlaR1, the β-lactam sensor/signal transducer protein, is phosphorylated on exposure to β-lactam antibiotics. This event is critical to the onset of the induction of antibiotic resistance. Furthermore, we document that BlaR1 phosphorylation and the antibiotic-resistance phenotype are both reversed in the presence of synthetic protein kinase inhibitors of our design, restoring susceptibility of the organism to a penicillin, resurrecting it from obsolescence in treatment of these intransigent bacteria.Keywords: BlaR1; kinase inhibitor; MRSA; phosphorylation; Stk1
Co-reporter:Mijoon Lee, Tomas Rucil, Dusan Hesek, Allen G. Oliver, Jed F. Fisher, and Shahriar Mobashery
The Journal of Organic Chemistry 2015 Volume 80(Issue 15) pp:7757-7763
Publication Date(Web):July 8, 2015
DOI:10.1021/acs.joc.5b01044
The SNAr reaction of 2,4-dichloropyrimidines, further substituted with an electron-withdrawing substituent at C-5, has selectivity for substitution at C-4. Here we report that tertiary amine nucleophiles show excellent C-2 selectivity. In situ N-dealkylation of an intermediate gives the product that formally corresponds to the reaction of a secondary amine nucleophile at C-2. This reaction is practical (fast under simple reaction conditions, with good generality for tertiary amine structure and moderate to excellent yields) and significantly expands access to pyrimidine structures.
Co-reporter:Mijoon Lee; Dusan Hesek; Blas Blázquez; Elena Lastochkin; Bill Boggess; Jed F. Fisher
Journal of the American Chemical Society 2014 Volume 137(Issue 1) pp:190-200
Publication Date(Web):December 11, 2014
DOI:10.1021/ja5111706
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen. A primary contributor to its ability to resist β-lactam antibiotics is the expression, following detection of the β-lactam, of the AmpC β-lactamase. As AmpC expression is directly linked to the recycling of the peptidoglycan of the bacterial cell wall, an important question is the identity of the signaling molecule(s) in this relationship. One mechanism used by clinical strains to elevate AmpC expression is loss of function of penicillin-binding protein 4 (PBP4). As the mechanism of the β-lactams is PBP inactivation, this result implies that the loss of the catalytic function of PBP4 ultimately leads to induction of antibiotic resistance. PBP4 is a bifunctional enzyme having both dd-carboxypeptidase and endopeptidase activities. Substrates for both the dd-carboxypeptidase and the 4,3-endopeptidase activities were prepared by multistep synthesis, and their turnover competence with respect to PBP4 was evaluated. The endopeptidase activity is specific to hydrolysis of 4,3-cross-linked peptidoglycan. PBP4 catalyzes both reactions equally well. When P. aeruginosa is grown in the presence of a strong inducer of AmpC, the quantities of both the stem pentapeptide (the substrate for the dd-carboxypeptidase activity) and the 4,3-cross-linked peptidoglycan (the substrate for the 4,3-endopeptidase activity) increase. In the presence of β-lactam antibiotics these altered cell-wall segments enter into the muropeptide recycling pathway, the conduit connecting the sensing event in the periplasm and the unleashing of resistance mechanisms in the cytoplasm.
Co-reporter:Peter I. O’Daniel ; Zhihong Peng ; Hualiang Pi ; Sebastian A. Testero ; Derong Ding ; Edward Spink ; Erika Leemans ; Marc A. Boudreau ; Takao Yamaguchi ; Valerie A. Schroeder ; William R. Wolter ; Leticia I. Llarrull ; Wei Song ; Elena Lastochkin ; Malika Kumarasiri ; Nuno T. Antunes ; Mana Espahbodi ; Katerina Lichtenwalter ; Mark A. Suckow ; Sergei Vakulenko ; Shahriar Mobashery ;Mayland Chang
Journal of the American Chemical Society 2014 Volume 136(Issue 9) pp:3664-3672
Publication Date(Web):February 11, 2014
DOI:10.1021/ja500053x
Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-β-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.
Co-reporter:Jennifer Fishovitz ; Alzoray Rojas-Altuve ; Lisandro H. Otero ; Matthew Dawley ; Cesar Carrasco-López ; Mayland Chang ; Juan A. Hermoso
Journal of the American Chemical Society 2014 Volume 136(Issue 28) pp:9814-9817
Publication Date(Web):June 23, 2014
DOI:10.1021/ja5030657
Ceftaroline, a recently approved β-lactam antibiotic for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.
Co-reporter:Major Gooyit, Zhihong Peng, William R. Wolter, Hualiang Pi, Derong Ding, Dusan Hesek, Mijoon Lee, Bill Boggess, Matthew M. Champion, Mark A. Suckow, Shahriar Mobashery, and Mayland Chang
ACS Chemical Biology 2014 Volume 9(Issue 1) pp:105
Publication Date(Web):September 20, 2013
DOI:10.1021/cb4005468
A complication of diabetes is the inability of wounds to heal in diabetic patients. Diabetic wounds are refractory to healing due to the involvement of activated matrix metalloproteinases (MMPs), which remodel the tissue resulting in apoptosis. There are no readily available methods that identify active unregulated MMPs. With the use of a novel inhibitor-tethered resin that binds exclusively to the active forms of MMPs, coupled with proteomics, we quantified MMP-8 and MMP-9 in a mouse model of diabetic wounds. Topical treatment with a selective MMP-9 inhibitor led to acceleration of wound healing, re-epithelialization, and significantly attenuated apoptosis. In contrast, selective pharmacological inhibition of MMP-8 delayed wound healing, decreased re-epithelialization, and exhibited high apoptosis. The MMP-9 activity makes the wounds refractory to healing, whereas that of MMP-8 is beneficial. The treatment of diabetic wounds with a selective MMP-9 inhibitor holds great promise in providing heretofore-unavailable opportunities for intervention of this disease.
Co-reporter:Cecilia Artola-Recolons, Mijoon Lee, Noelia Bernardo-García, Blas Blázquez, Dusan Hesek, Sergio G. Bartual, Kiran V. Mahasenan, Elena Lastochkin, Hualiang Pi, Bill Boggess, Kathrin Meindl, Isabel Usón, Jed F. Fisher, Shahriar Mobashery, and Juan A. Hermoso
ACS Chemical Biology 2014 Volume 9(Issue 9) pp:2058
Publication Date(Web):July 2, 2014
DOI:10.1021/cb500439c
The lytic transglycosylases are essential bacterial enzymes that catalyze the nonhydrolytic cleavage of the glycan strands of the bacterial cell wall. We describe here the structural and catalytic properties of MltC, one of the seven lytic transglycosylases found in the genome of the Gram-negative bacterium Escherichia coli. The 2.3 Å resolution X-ray structure of a soluble construct of MltC shows a unique, compared to known lytic transglycosylase structures, two-domain structure characterized by an expansive active site of 53 Å length extending through an interface between the domains. The structures of three complexes of MltC with cell wall analogues suggest the positioning of the peptidoglycan in the active site both as a substrate and as a product. One complex is suggested to correspond to an intermediate in the course of sequential and exolytic cleavage of the peptidoglycan. Moreover, MltC partitioned its reactive oxocarbenium-like intermediate between trapping by the C6-hydroxyl of the muramyl moiety (lytic transglycosylase activity, the major path) and by water (muramidase activity). Genomic analysis identifies the presence of an MltC homologue in no less than 791 bacterial genomes. While the role of MltC in cell wall assembly and maturation remains uncertain, we propose a functional role for this enzyme as befits the uniqueness of its two-domain structure.
Co-reporter:Blas Blázquez, Leticia I. Llarrull, Juan R. Luque-Ortega, Carlos Alfonso, Bill Boggess, and Shahriar Mobashery
Biochemistry 2014 Volume 53(Issue 10) pp:
Publication Date(Web):February 24, 2014
DOI:10.1021/bi500074w
β-Lactam antibiotics have faced obsolescence with the emergence of methicillin-resistant Staphylococcus aureus (MRSA). A complex set of events ensues upon exposure of MRSA to these antibiotics, which culminates in proteolysis of BlaI or MecI, two gene repressors, and results in the induction of resistance. We report studies on the mechanism of binding of these gene repressors to the operator regions by fluorescence anisotropy. Within the range of in vivo concentrations for BlaI and MecI, these proteins interact with their regulatory elements in a reversible manner, as both a monomer and a dimer.
Co-reporter:Derong Ding, Katerina Lichtenwalter, Hualiang Pi, Shahriar Mobashery and Mayland Chang
MedChemComm 2014 vol. 5(Issue 9) pp:1381-1383
Publication Date(Web):18 Jun 2014
DOI:10.1039/C4MD00172A
MMP-8 has been implicated in various diseases. Selective MMP-8 inhibitors are needed to ascertain the role of this enzyme. We synthesized two inhibitors reported previously as selective for MMP-8. Compound 1 selectively inhibited MMP-8 and MMP-13; compound 2 was a potent broad-spectrum inhibitor, notwithstanding that it is used as a selective one.
Co-reporter:Jed F. Fisher, Shahriar Mobashery
Bioorganic Chemistry 2014 Volume 55() pp:78
Publication Date(Web):August 2014
DOI:10.1016/j.bioorg.2014.07.006
Co-reporter:Jed F. Fisher, Shahriar Mobashery
Bioorganic Chemistry 2014 Volume 56() pp:41-48
Publication Date(Web):October 2014
DOI:10.1016/j.bioorg.2014.05.011
•Gram-negative bacterial infections often are treated with β-lactam antibiotics.•β-Lactam antibiotics target the peptidoglycan polymer of the bacterium.•The peptidoglycan of Gram-negative bacteria is continuously remodeled.•Bacteria sense the presence of β-lactams by their effect on peptidoglycan remodeling.•Blocking this signaling pathway may subvert bacterial antibiotic resistance.The peptidoglycan is the structural polymer of the bacterial cell envelope. In contrast to an expectation of a structural stasis for this polymer, during the growth of the Gram-negative bacterium this polymer is in a constant state of remodeling and extension. Our current understanding of this peptidoglycan “turnover” intertwines with the deeply related phenomena of the liberation of small peptidoglycan segments (muropeptides) during turnover, the presence of dedicated recycling pathways for reuse of these muropeptides, β-lactam inactivation of specific penicillin-binding proteins as a mechanism for the perturbation of the muropeptide pool, and this perturbation as a controlling mechanism for signal transduction leading to the expression of β-lactamase(s) as a key resistance mechanism against the β-lactam antibiotics. The nexus for many of these events is the control of the AmpR transcription factor by the composition of the muropeptide pool generated during peptidoglycan recycling. In this review we connect the seminal observations of the past decades to new observations that resolve some, but certainly not all, of the key structures and mechanisms that connect to AmpR.
Co-reporter:Mijoon Lee;Dusan Hesek;Bruce C. Noll;Allen G. Oliver
Acta Crystallographica Section C 2014 Volume 70( Issue 11) pp:1003-1006
Publication Date(Web):
DOI:10.1107/S2053229614021214
The compound 2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane, C15H14O3S2, a selective gelatinase inhibitor, was synthesized and structurally characterized. Two crystals were analyzed, one each for the R and S enantiomers, and the results were compared with the previously reported structure of the racemate. The enantiomerically pure compounds both crystallize with Z′ = 2 in the space group P21, while the racemic mixture crystallizes with Z′ = 1 in the space group P21/c, with disorder in the position of the thiirane group. This disorder accommodates both molecules for each of the enantiomerically pure crystals, showing good overlap of the molecules of the pure enantiomorphs with the components of the centrosymmetric structure.
Co-reporter:Mijoon Lee ; Dusan Hesek ; Leticia I. Llarrull ; Elena Lastochkin ; Hualiang Pi ; Bill Boggess
Journal of the American Chemical Society 2013 Volume 135(Issue 9) pp:3311-3314
Publication Date(Web):February 19, 2013
DOI:10.1021/ja309036q
The reactions of all seven Escherichia coli lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall.
Co-reporter:Weilie Zhang ; Mijoon Lee ; Dusan Hesek ; Elena Lastochkin ; Bill Boggess
Journal of the American Chemical Society 2013 Volume 135(Issue 13) pp:4950-4953
Publication Date(Web):March 19, 2013
DOI:10.1021/ja400970n
A group of Gram-negative bacteria, including the problematic pathogen Pseudomonas aeruginosa, has linked the steps in cell-wall recycling with the ability to manifest resistance to β-lactam antibiotics. A key step at the crossroads of the two events is performed by the protease AmpD, which hydrolyzes the peptide in the metabolite that influences these events. In contrast to other organisms that harbor this elaborate system, the genomic sequences of P. aeruginosa reveal it to have three paralogous genes for this protease, designated as ampD, ampDh2, and ampDh3. The recombinant gene products were purified to homogeneity, and their functions were assessed by the use of synthetic samples of three bacterial metabolites in cell-wall recycling and of three surrogates of cell-wall peptidoglycan. The results unequivocally identify AmpD as the bona fide recycling enzyme and AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself. These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation.
Co-reporter:Siseth Martínez-Caballero ; Mijoon Lee ; Cecilia Artola-Recolons ; César Carrasco-López ; Dusan Hesek ; Edward Spink ; Elena Lastochkin ; Weilie Zhang ; Lance M. Hellman ; Bill Boggess ; Shahriar Mobashery ;Juan A. Hermoso
Journal of the American Chemical Society 2013 Volume 135(Issue 28) pp:10318-10321
Publication Date(Web):July 2, 2013
DOI:10.1021/ja405464b
The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall.
Co-reporter:Mijoon Lee ; Cecilia Artola-Recolons ; César Carrasco-López ; Siseth Martínez-Caballero ; Dusan Hesek ; Edward Spink ; Elena Lastochkin ; Weilie Zhang ; Lance M. Hellman ; Bill Boggess ; Juan A. Hermoso
Journal of the American Chemical Society 2013 Volume 135(Issue 34) pp:12604-12607
Publication Date(Web):August 9, 2013
DOI:10.1021/ja407445x
Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa, which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling.
Co-reporter:Lisandro H. Otero;Malika Kumarasiri;Alzoray Rojas-Altuve;Elena Lastochkin;Cesar Carrasco-López;Mijoon Lee;Leticia I. Llarrull;Mayland Chang;Matthew Dawley;Dusan Hesek;Jed F. Fisher;Juan A. Hermoso;Jennifer Fishovitz;Jarrod W. Johnson
PNAS 2013 Volume 110 (Issue 42 ) pp:16808-16813
Publication Date(Web):2013-10-15
DOI:10.1073/pnas.1300118110
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics
by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase
and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell
wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs)
is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a
of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics
is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60
Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric
site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry.
This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component
of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The
ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to
inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.
Co-reporter:Lukas Jedinak, LaToya Rush, Mijoon Lee, Dusan Hesek, Jed F. Fisher, Bill Boggess, Bruce C. Noll, and Shahriar Mobashery
The Journal of Organic Chemistry 2013 Volume 78(Issue 23) pp:12224-12228
Publication Date(Web):November 19, 2013
DOI:10.1021/jo401972a
An efficient synthesis of olefins by the coupling of stabilized, semistabilized, and nonstabilized phosphorus ylides with various carbonyl compounds in the presence of silver carbonate is reported. Wittig olefination of aromatic, heteroaromatic, and aliphatic aldehydes (yields >63%) and a ketone (yield 42%) are demonstrated. These reactions proceed overnight at room temperature, under weakly basic conditions, and as such extend the applicability of the Wittig reaction to base-sensitive reactants.
Co-reporter:Dusan Hesek ; Mijoon Lee ; Jaroslav Zajíček ; Jed F. Fisher
Journal of the American Chemical Society 2012 Volume 134(Issue 33) pp:13881-13888
Publication Date(Web):August 4, 2012
DOI:10.1021/ja306184m
We describe a practical, multigram synthesis of (2Z,6Z,10Z,14Z,18E,22E)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(Z4,E2,ω)-heptaprenol, 4] using the nerol-derived sulfone 8 as the key intermediate. Sulfone 8 is prepared by the literature route and is converted in five additional steps (18% yield from 8) to (Z4,E2,ω)-heptaprenol 4. The use of Eu(hfc)3 as an NMR shift reagent not only enabled confirmation of the structure and stereochemistry of 4, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (Z4,E2,ω)-heptaprenol 4 in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope.
Co-reporter:Takao Yamaguchi, Blas Blázquez, Dusan Hesek, Mijoon Lee, Leticia I. Llarrull, Bill Boggess, Allen G. Oliver, Jed F. Fisher, and Shahriar Mobashery
ACS Medicinal Chemistry Letters 2012 Volume 3(Issue 3) pp:238
Publication Date(Web):January 19, 2012
DOI:10.1021/ml2002746
Gram-negative bacteria have evolved an elaborate process for the recycling of their cell wall, which is initiated in the periplasmic space by the action of lytic transglycosylases. The product of this reaction, β-d-N-acetylglucosamine-(1→4)-1,6-anhydro-β-d-N-acetylmuramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala (compound 1), is internalized to begin the recycling events within the cytoplasm. The first step in the cytoplasmic recycling is catalyzed by the NagZ glycosylase, which cleaves in a hydrolytic reaction the N-acetylglucosamine glycosidic bond of metabolite 1. The reactions catalyzed by both the lytic glycosylases and NagZ are believed to involve oxocarbenium transition species. We describe herein the synthesis and evaluation of four iminosaccharides as possible mimetics of the oxocarbenium species, and we disclose one as a potent (compound 3, Ki = 300 ± 15 nM) competitive inhibitor of NagZ.Keywords: Iminosaccharide; MltB; NagZ; peptidoglycan; β-lactam
Co-reporter:Marc A. Boudreau, Jed F. Fisher, and Shahriar Mobashery
Biochemistry 2012 Volume 51(Issue 14) pp:2974-2990
Publication Date(Web):March 12, 2012
DOI:10.1021/bi300174x
Bacterial muropeptides are soluble peptidoglycan structures central to recycling of the bacterial cell wall and messengers in diverse cell signaling events. Bacteria sense muropeptides as signals that antibiotics targeting cell-wall biosynthesis are present, and eukaryotes detect muropeptides during the innate immune response to bacterial infection. This review summarizes the roles of bacterial muropeptides as messengers, with a special emphasis on bacterial muropeptide structures and the relationship of structure to the biochemical events that the muropeptides elicit. Muropeptide sensing and recycling in both Gram-positive and Gram-negative bacteria are discussed, followed by muropeptide sensing by eukaryotes as a crucial event in the innate immune response of insects (via peptidoglycan-recognition proteins) and mammals (through Nod-like receptors) to bacterial invasion.
Co-reporter:Leticia I. Llarrull and Shahriar Mobashery
Biochemistry 2012 Volume 51(Issue 23) pp:4642-4649
Publication Date(Web):May 22, 2012
DOI:10.1021/bi300429p
A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.
Co-reporter:Qicun Shi ; Samy O. Meroueh ; Jed F. Fisher
Journal of the American Chemical Society 2011 Volume 133(Issue 14) pp:5274-5283
Publication Date(Web):March 18, 2011
DOI:10.1021/ja1074739
Penicillin-binding protein 1b (PBP 1b) of the Gram-positive bacterium Streptococcus pneumoniae catalyzes the cross-linking of adjacent peptidoglycan strands, as a critical event in the biosynthesis of its cell wall. This enzyme is representative of the biosynthetic PBP structures of the β-lactam-recognizing enzyme superfamily and is the target of the β-lactam antibiotics. In the cross-linking reaction, the amide between the -d-Ala-d-Ala dipeptide at the terminus of a peptide stem acts as an acyl donor toward the ε-amino group of a lysine found on an adjacent stem. The mechanism of this transpeptidation was evaluated using explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics calculations. Sequential acyl transfer occurs to, and then from, the active site serine. The resulting cross-link is predicted to have a cis-amide configuration. The ensuing and energetically favorable cis- to trans-amide isomerization, within the active site, may represent the key event driving product release to complete enzymatic turnover.
Co-reporter:Sebastian A. Testero, Mijoon Lee, Rachel T. Staran, Mana Espahbodi, Leticia I. Llarrull, Marta Toth, Shahriar Mobashery, and Mayland Chang
ACS Medicinal Chemistry Letters 2011 Volume 2(Issue 2) pp:177
Publication Date(Web):December 13, 2010
DOI:10.1021/ml100254e
Matrix metalloproteinases (MMPs) are important zinc-dependent endopeptidases. Two members of this family of enzymes called gelatinases (MMP-2 and MMP-9) have been implicated in a number of human diseases, including cancer, neurological and cardiovascular diseases, and inflammation, to name a few. We describe in this report the preparation and evaluation of two structural types of thiirane inhibitors that show selectivity toward gelatinases. The biphenyl series targets both gelatinases, whereas the monophenyl analogues exhibit potent inhibition of only MMP-2. The latter structural type also exhibits improved water solubility and metabolic stability, both traits desirable for progress of these molecules forward in gelatinase-dependent animal models of disease.Keywords (keywords): matrix metalloproteinases; Slow-binding gelatinase inhibitors; thiiranes
Co-reporter:Sebastian A. Testero, Renee Bouley, Jed F. Fisher, Mayland Chang, Shahriar Mobashery
Bioorganic & Medicinal Chemistry Letters 2011 21(9) pp: 2675-2678
Publication Date(Web):
DOI:10.1016/j.bmcl.2010.12.076
Co-reporter:Jed F. Fisher
Journal of Medicinal Chemistry 2010 Volume 53(Issue 13) pp:4813-4829
Publication Date(Web):June 4, 2010
DOI:10.1021/jm100086u
Co-reporter:Leticia I. Llarrull, Mary Prorok, and Shahriar Mobashery
Biochemistry 2010 Volume 49(Issue 37) pp:
Publication Date(Web):August 19, 2010
DOI:10.1021/bi101177a
The expression of the gene products in many methicillin-resistant Staphylococcus aureus (MRSA) strains is regulated by the gene repressor BlaI. Here we show that BlaI is a mixture of monomer and dimer at in vivo concentrations, binds to the operator regions preferentially as a monomeric protein, and the measured dissociation constants and in vivo concentrations account for the basal level transcription of the resistance genes. These observations for the first time provide a quantitative picture of the processes that take place in the cytoplasm that lead to the induction of antibiotic resistance factors to counter the challenge by β-lactams.
Co-reporter:Daniel Fernández;Sebastian Testero;Josep Vendrell;Francesc X. Avilés
Chemical Biology & Drug Design 2010 Volume 75( Issue 1) pp:29-34
Publication Date(Web):
DOI:10.1111/j.1747-0285.2009.00907.x
The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 Å resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1’ specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.
Co-reporter:Dr. Mijoon Lee;Dr. Dusan Hesek;Dr. Ishita M. Shah;Dr. Allen G. Oliver;Dr. Jonathan Dworkin;Dr. Shahriar Mobashery
ChemBioChem 2010 Volume 11( Issue 18) pp:2525-2529
Publication Date(Web):
DOI:10.1002/cbic.201000626
Co-reporter:Mijoon Lee ; Weilie Zhang ; Dusan Hesek ; Bruce C. Noll ; Bill Boggess
Journal of the American Chemical Society 2009 Volume 131(Issue 25) pp:8742-8743
Publication Date(Web):June 4, 2009
DOI:10.1021/ja9025566
The bacterial enzyme AmpD is an early catalyst in commitment of cell wall metabolites to the recycling events within the cytoplasm. The key internalized metabolite of cell wall recycling, β-D-N-acetylglucosamine-(1→4)-1,6-anhydro-β-N-acetylmuramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala (compound 1), is a poor substrate for AmpD. Two additional metabolites, 1,6-anhydro-N-acetylmuramyl-peptidyl derivatives 2a and 2c, served as substrates for AmpD with a kcat/Km of >104 M−1 s−1. The enzyme hydrolytically processes the lactyl amide bond of the 1,6-anhydro-N-acetylmuramyl moiety. The syntheses of these substrates and other ligands are reported herein, which made the characterization of the enzymic reaction possible. Furthermore, it is documented that the enzyme is specific for both the atypical peptide stem of the cell wall fragments and the presence of the sterically encumbered 1,6-anhydro-N-acetylmuramyl moiety; hence it is a peptidase with a unique function in bacterial physiology. The implications of the function of this catalyst for the entry into the cell wall recycling events and the reversal of induction of the production of β-lactamase, an antibiotic resistance determinant, are discussed.
Co-reporter:Yu Chen ; Weilie Zhang ; Qicun Shi ; Dusan Hesek ; Mijoon Lee ; Shahriar Mobashery ;Brian K. Shoichet
Journal of the American Chemical Society 2009 Volume 131(Issue 40) pp:14345-14354
Publication Date(Web):September 17, 2009
DOI:10.1021/ja903773f
Penicillin-binding protein 6 (PBP6) is one of the two main dd-carboxypeptidases in Escherichia coli, which are implicated in maturation of bacterial cell wall and formation of cell shape. Here, we report the first X-ray crystal structures of PBP6, capturing its apo state (2.1 Å), an acyl-enzyme intermediate with the antibiotic ampicillin (1.8 Å), and for the first time for a PBP, a preacylation complex (a “Michaelis complex”, determined at 1.8 Å) with a peptidoglycan substrate fragment containing the full pentapeptide, NAM-(l-Ala-d-isoGlu-l-Lys-d-Ala-d-Ala). These structures illuminate the molecular interactions essential for ligand recognition and catalysis by dd-carboxypeptidases, and suggest a coupling of conformational flexibility of active site loops to the reaction coordinate. The substrate fragment complex structure, in particular, provides templates for models of cell wall recognition by PBPs, as well as substantiating evidence for the molecular mimicry by β-lactam antibiotics of the peptidoglycan acyl-d-Ala-d-Ala moiety.
Co-reporter:Sebastian A. Testero, Peter I. O’Daniel, Qicun Shi, Mijoon Lee, Dusan Hesek, Akihiro Ishiwata, Bruce C. Noll and Shahriar Mobashery
Organic Letters 2009 Volume 11(Issue 12) pp:2515-2518
Publication Date(Web):May 15, 2009
DOI:10.1021/ol900668k
The unique hydrophobic surface patches in class D β-lactamases presented an opportunity for designing two compounds, 6α-(1R-hydroxyoctyl)penicillanic acid and 6β-(1R-hydroxyoctyl)penicillanic acid, as mechanistic probes of these enzymes. In a sequence of three synthetic steps from benzhydryl 6,6-dibromopenicillanate, the targeted compounds were prepared in a stereospecific manner.
Co-reporter:Major Gooyit;Mijoon Lee;Dusan Hesek;Bill Boggess;Allen G. Oliver;Rafael Fridman;Mayl Chang
Chemical Biology & Drug Design 2009 Volume 74( Issue 6) pp:535-546
Publication Date(Web):
DOI:10.1111/j.1747-0285.2009.00898.x
Gelatinases (MMP-2 and MMP-9) have been implicated in a number of pathological conditions, including cancer and cardiovascular disease. Hence, small molecule inhibitors of these enzymes are highly sought for use as potential therapeutic agents. 2-(4-Phenoxyphenylsulfonylmethyl)thiirane (SB-3CT) has previously been demonstrated to be a potent and selective inhibitor of gelatinases, however, it is rapidly metabolized because of oxidation at the para position of the phenoxy ring and at the α-position to the sulfonyl group. α-Methyl variants of SB-3CT were conceived to improve metabolic stability and as mechanistic probes. We describe herein the synthesis and evaluation of these structural variants as potent inhibitors of gelatinases. Two (compounds 5b and 5d) among the four synthetic stereoisomers were found to exhibit slow-binding inhibition of gelatinases and MMP-14 (MT1-MMP), which is a hallmark of the mechanism of this class of inhibitors. The ability of these compounds to inhibit MMP-2, MMP-9, and MMP-14 could target cancer tissues more effectively. Metabolism of the newly synthesized inhibitors showed that both oxidation at the α-position to the sulfonyl group and oxidation at the para position of the terminal phenyl ring were prevented. Instead oxidation on the thiirane sulfur is the only biotransformation pathway observed for these gelatinase inhibitors.
Co-reporter:Christopher Forbes;Qicun Shi; Jed F. Fisher;Mijoon Lee;Dusan Hesek;Leticia I. Llarrull;Marta Toth; Michael Gossing;Rafael Fridman
Chemical Biology & Drug Design 2009 Volume 74( Issue 6) pp:527-534
Publication Date(Web):
DOI:10.1111/j.1747-0285.2009.00881.x
(±)-2-[(4-Phenoxyphenylsulfonyl)methyl]thiirane 1 is a potent and selective mechanism-based inhibitor of the gelatinase sub-class of the zinc-dependent matrix metalloproteinase family. Inhibitor 1 has excellent activity in in vivo models of gelatinase-dependent disease. We demonstrate that the mechanism of inhibition is a rate-limiting gelatinase-catalyzed thiolate generation via deprotonation adjacent to the thiirane, with concomitant thiirane opening. A corollary to this mechanism is the prediction that thiol-containing structures, related to thiirane-opened 1, will possess potent matrix metalloproteinase inhibitory activity. This prediction was validated by the synthesis of the product of this enzyme-catalyzed reaction on 1, which exhibited a remarkable Ki of 530 pm against matrix metalloproteinase-2. Thiirane 1 acts as a caged thiol, unmasked selectively in the active sites of gelatinases. This mechanism is unprecedented in the substantial literature on inhibition of zinc-dependent hydrolases.
Co-reporter:Peng Tao, Jed F. Fisher, Qicun Shi, Thom Vreven, Shahriar Mobashery and H. Bernhard Schlegel
Biochemistry 2009 Volume 48(Issue 41) pp:
Publication Date(Web):September 15, 2009
DOI:10.1021/bi901118r
The inhibition mechanism of matrix metalloproteinase 2 (MMP2) by the selective inhibitor (4-phenoxyphenylsulfonyl)methylthiirane (SB-3CT) and its oxirane analogue is investigated computationally. The inhibition mechanism involves C−H deprotonation with concomitant opening of the three-membered heterocycle. SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part and the AMBER force field for the MM part were used to examine the reaction of these two inhibitors in the active site of MMP2. The calculations show that the reaction barrier for transformation of SB-3CT is 1.6 kcal/mol lower than its oxirane analogue, and the ring-opening reaction energy of SB-3CT is 8.0 kcal/mol more exothermic than that of its oxirane analogue. Calculations also show that protonation of the ring-opened product by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane than for the thiolate obtained from the thiirane. A six-step partial charge fitting procedure is introduced for the QM/MM calculations to update atomic partial charges of the quantum mechanics region and to ensure consistent electrostatic energies for reactants, transition states, and products.
Co-reporter:Mijoon Lee;Giuseppe Celenza;Bill Boggess;Jennifer Blase;Qicun Shi;Marta Toth;M. Margarida Bernardo;William R. Wolter;Mark A. Suckow;Dusan Hesek;Bruce C. Noll;Rafael Fridman;Mayl Chang
Chemical Biology & Drug Design 2009 Volume 73( Issue 2) pp:189-202
Publication Date(Web):
DOI:10.1111/j.1747-0285.2008.00750.x
Metastatic tumors lead to more than 90% fatality. Despite the importance of invasiveness of tumors to poor disease outcome, no anti-invasive compounds have been commercialized. We describe herein the synthesis and evaluation of 4-(4-(thiiranylmethylsulfonyl)phenoxy)-phenyl methanesulfonate (compound 2) as a potent and selective inhibitor of gelatinases (matrix metalloproteinases-2 and -9), two enzymes implicated in invasiveness of tumors. It was demonstrated that compound 2 significantly attenuated the invasiveness of human fibrosarcoma cells (HT1080). The metabolism of compound 2 involved hydroxylation at the α-methylene, which generates sulfinic acid, thiirane ring-opening, followed by methylation and oxidation, and cysteine conjugation of both the thiirane and phenyl rings.
Co-reporter:Mijoon Lee;Dusan Hesek;Bruce C. Noll
Chemical Papers 2009 Volume 63( Issue 5) pp:592-597
Publication Date(Web):2009 October
DOI:10.2478/s11696-009-0048-0
Dimethylmaleoyl (DMM) moiety has become an important amine protective group in sugar chemistry. We disclose herein that DMM-containing D-glucosamine analogues, because of their electrophilic nature, are prone to reactions with strong nucleophiles, such as hydrazine, resulting in a set of undesired side products that are difficult to detect, yet proved to be problematic for organic synthesis.
Co-reporter:Samy O. Meroueh
Chemical Biology & Drug Design 2007 Volume 69(Issue 5) pp:
Publication Date(Web):17 MAY 2007
DOI:10.1111/j.1747-0285.2007.00505.x
Peptide bonds are made at the ribosomal decoding site. Structural information reveals that two bases in the RNA that constitute the decoding site, A1492 and A1493, can have both intrahelical and extrahelical conformations. Aminoglycoside antibiotics bind to the decoding site, and the structural information reveals the two bases in the extrahelical positions. We have shown by explicit-solvent molecular dynamics simulations and free-energy calculations that ribosomal RNA bases A1492 and A1493 are inherently prone to sampling conformational states that include both intrahelical and extrahelical positions. The simulations reveal that base flipping occurs through the minor groove of the double helix. Furthermore, free-energy calculations for the conformational change of the bases to the extrahelical positions in both processes are exergonic and highly favorable. It is likely that the correct codon-anticodon recognition by mRNA and tRNA arrests the bases in extrahelical conformations in the course of normal translation. In contrast, the sequestration of the aminoglycoside antibiotic at the decoding site facilitates the conformational change of the bases to the extrahelical position. Once the antibiotic is bound, the extrahelical positions for the bases are highly favored based on contributions by both electrostatic and entropic components of the free energy for the process.
Co-reporter:Sangwoo Cho;Qian Wang;Mijoon Lee;Geert-Jan Boons;Roy A. Mariuzza;Chittoor P. Swaminathan;Dusan Hesek
PNAS 2007 Volume 104 (Issue 21 ) pp:8761-8766
Publication Date(Web):2007-05-22
DOI:10.1073/pnas.0701453104
Peptidoglycan recognition proteins (PGRPs) are highly conserved pattern-recognition molecules of the innate immune system
that bind bacterial peptidoglycans (PGNs), which are polymers of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by short peptide stems. Human PRGPs are bactericidal against pathogenic and nonpathogenic
Gram-positive bacteria, but not normal flora bacteria. Like certain glycopeptide antibiotics (e.g., vancomycin), PGRPs kill
bacteria by directly interacting with their cell wall PGN, thereby interfering with PGN maturation. To better understand the
bactericidal mechanism of PGRPs, we determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Iβ
in complex with NAG-NAM-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala, a synthetic glycopeptide comprising a complete PGN repeat. This structure, in conjunction with the previously reported
NMR structure of a dimeric PGN fragment, permitted identification of major conformational differences between free and PGRP-bound
PGN with respect to the relative orientation of saccharide and peptide moieties. These differences provided structural insights
into the bactericidal mechanism of human PGRPs. On the basis of molecular modeling, we propose that these proteins disrupt
cell wall maturation not only by sterically encumbering access of biosynthetic enzymes to the nascent PGN chains, but also
by locking PGN into a conformation that prevents formation of cross-links between peptide stems in the growing cell wall.
Co-reporter:Dusan Hesek, Marta Toth, Samy O. Meroueh, Stephen Brown, Huiren Zhao, Wael Sakr, Rafael Fridman, Shahriar Mobashery
Chemistry & Biology 2006 Volume 13(Issue 4) pp:379-386
Publication Date(Web):April 2006
DOI:10.1016/j.chembiol.2006.01.012
Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, are implicated in tumor progression. We describe herein the development of a resin-immobilized, broad-spectrum synthetic MMP inhibitor for selective binding of the active forms of MMPs from different experimental samples. We confirmed the activity-based binding of MMPs to the inhibitor-tethered resin with purified human recombinant MMP-2, -9, and -14, samples of cultured cells, and tissue extracts. Our results show that only the free active MMPs, and not the zymogens or MMP/TIMP (enzyme-protein inhibitor) complexes, bound specifically to the resin. In our comparison of benign and carcinoma tissue extracts, we detected active MMP-2 and MMP-14 forms only in the cancerous tissue samples, indicating that a pool of the tumor MMPs is free of endogenous inhibitors (TIMPs), and is thus likely to contribute to proteolytic events that precipitate tumor metastasis.
Co-reporter:James B. Murray, Samy O. Meroueh, Rupert J.M. Russell, Georg Lentzen, Jalal Haddad, Shahriar Mobashery
Chemistry & Biology 2006 Volume 13(Issue 2) pp:129-138
Publication Date(Web):February 2006
DOI:10.1016/j.chembiol.2005.11.004
The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5–3.0 Å resolution and that of neamine at 2.8 Å resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Å resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.
Co-reporter:Choonkeun Kim, Shahriar Mobashery
Bioorganic Chemistry 2005 Volume 33(Issue 3) pp:149-158
Publication Date(Web):June 2005
DOI:10.1016/j.bioorg.2004.11.001
Transfer of the γ-phosphoryl group from ATP to aminoglycoside antibiotics by aminoglycoside 3′-phosphotransferases is one of the most important reactions for manifestation of bacterial resistance to this class of antibiotics. This review article surveys the latest structural and mechanistic findings with these enzymes.
Co-reporter:Mijoon Lee, Rafael Fridman and Shahriar Mobashery
Chemical Society Reviews 2004 vol. 33(Issue 7) pp:401-409
Publication Date(Web):13 Aug 2004
DOI:10.1039/B209224G
Metastasis, the dissemination of tumor cells to distant organs, is often associated with fatal outcome in cancer patients. Formation of metastasis requires degradation of extracellular matrices and several families of proteases have been implicated in this process, including matrix metalloproteinases (MMPs), serine and cysteine proteases. Inhibition of these enzymes in animal models of metastasis has shown impressive therapeutic effects. This report discusses the various approaches used for enzyme inhibition and describes new developments in drug design for inhibition of proteases in metastatic disease.
Co-reporter:Jeshina Janardhanan, Mayland Chang, Shahriar Mobashery
Current Opinion in Microbiology (October 2016) Volume 33() pp:13-17
Publication Date(Web):1 October 2016
DOI:10.1016/j.mib.2016.05.009
The oxadiazoles are a class of antibacterials discovered by in silico docking and scoring of compounds against the X-ray structure of a penicillin-binding protein. These antibacterials exhibit activity against Gram-positive bacteria, including against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). They show in vivo efficacy in murine models of peritonitis/sepsis and neutropenic thigh MRSA infection. They are bactericidal and orally bioavailable. The oxadiazoles show promise in treatment of MRSA infection.
Co-reporter:Teresa Domínguez-Gil, Mijoon Lee, Iván Acebrón-Avalos, Kiran V. Mahasenan, ... Juan A. Hermoso
Structure (4 October 2016) Volume 24(Issue 10) pp:1729-1741
Publication Date(Web):4 October 2016
DOI:10.1016/j.str.2016.07.019
•Modular LT having a catalytic module and an ABC-transporter-like regulatory module•Both regulatory module and the linker region keep MltF in an inactive conformation•Muropeptide binding at regulatory module triggers MltF activation•First case of allosteric regulation in an LT spurred by dramatic structural changesBacteria grow and divide without loss of cellular integrity. This accomplishment is notable, as a key component of their cell envelope is a surrounding glycopeptide polymer. In Gram-negative bacteria this polymer—the peptidoglycan—grows by the difference between concurrent synthesis and degradation. The regulation of the enzymatic ensemble for these activities is poorly understood. We report herein the structural basis for the control of one such enzyme, the lytic transglycosylase MltF of Pseudomonas aeruginosa. Its structure comprises two modules: an ABC-transporter-like regulatory module and a catalytic module. Occupancy of the regulatory module by peptidoglycan-derived muropeptides effects a dramatic and long-distance (40 Å) conformational change, occurring over the entire protein structure, to open its active site for catalysis. This discovery of the molecular basis for the allosteric control of MltF catalysis is foundational to further study of MltF within the complex enzymatic orchestration of the dynamic peptidoglycan.Download high-res image (246KB)Download full-size image
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