New synthetic routes to Triazolo-benzodiazepine analogues:expanding the scope of the bump-and-hole approach for selective Bromo and Extra-Terminal (BET) bromodomain inhibition

We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra-terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl rings, in addition to the diazepine ternary center and the side chain methylene moiety. Substitution at the level of the side chain methylene afforded compounds targeting specifically and potently engineered BET bromodomains designed as part of a bump and hole approach. We further demonstrate that marked selectivity for the second over the first bromodomain can be achieved with an indole derivative that exploits differential interaction with an aspartate/histidine conservative substitution on the BC loop of BET bromodomains

Activity of ulilysin, an archaeal PAPP-A-related gelatinase and IGFBP protease

Human growth and development are conditioned by insulin-like growth factors (IGFs), which have also implications in pathology. Most IGF molecules are sequestered by IGF-binding proteins (IGFBPs) so that exertion of IGF activity requires disturbance of these complexes. This is achieved by proteolysis mediated by IGFBP proteases, among which the best characterised is human PAPP-A, the first member of the pappalysin family of metzincins. We have previously identified and studied the only archaeal homologue found to date, Methanosarcina acetivorans ulilysin. This is a proteolytically functional enzyme encompassing a pappalysin catalytic domain and a pro-domain involved in maintenance of latency of the zymogen, proulilysin. Once activated, the protein hydrolyses IGFBP-2 to -6 and insulin chain β in vitro. We report here that ulilysin is also active against several other substrates, viz (azo)casein, azoalbumin, and extracellular matrix components. Ulilysin has gelatinolytic but not collagenolytic activity. Moreover, the proteolysis-resistant skeletal proteins actin and elastin are also cleaved, as is fibrinogen, but not plasmin and α1-antitrypsin from the blood coagulation cascade. Ulilysin develops optimal activity at pH 7.5 and strictly requires peptide bonds preceding an arginine residue, as determined by means of a novel fluorescence resonance energy transfer assay, thus pointing to biotechnological applications as an enzyme complementary to trypsi

Discovery and optimization of a selective ligand for the switch/sucrose nonfermenting-related bromodomains of polybromo protein-1 by the use of virtual screening and hydration analysis

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein−ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5)

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

El pdf es el manuscrito de autor.-- et al.Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1+, pXO2 -), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1 + A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture. © 2011 Elsevier Inc. All rights reserved.This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA.Peer Reviewe

Matrix metalloproteinases: Fold and function of their catalytic domains

Matrix metalloproteinases (MMPs) are zinc-dependent protein and peptide hydrolases. They have been almost exclusively studied in vertebrates and 23 paralogs are present in humans. They are widely involved in metabolism regulation through both extensive protein degradation and selective peptide-bond hydrolysis. If MMPs are not subjected to exquisite spatial and temporal control, they become destructive, which can lead to pathologies such as arthritis, inflammation, and cancer. The main therapeutic strategy to combat the dysregulation of MMPs is the design of drugs to target their catalytic domains, for which purpose detailed structural knowledge is essential. The catalytic domains of 13 MMPs have been structurally analyzed so far and they belong to the >metzincin> clan of metalloendopeptidases. These compact, spherical, ~165-residue molecules are divided by a shallow substrate-binding crevice into an upper and a lower sub-domain. The molecules have an extended zinc-binding motif, HEXXHXXGXXH, which contains three zinc-binding histidines and a glutamate that acts as a general base/acid during catalysis. In addition, a conserved methionine lying within a >Met-turn> provides a hydrophobic base for the zinc-binding site. Further earmarks of MMPs are three α-helices and a five-stranded β-sheet, as well as at least two calcium sites and a second zinc site with structural functions. Most MMPs are secreted as inactive zymogens with an N-terminal ~80-residue pro-domain, which folds into a three-helix globular domain and inhibits the catalytic zinc through a cysteine imbedded in a conserved motif, PRCGXPD. Removal of the pro-domain enables access of a catalytic solvent molecule and substrate molecules to the active-site cleft, which harbors a hydrophobic S1&core;-pocket as main determinant of specificity. Together with the catalytic zinc ion, this pocket has been targeted since the onset of drug development against MMPs. However, the inability of first- and second-generation inhibitors to distinguish between different MMPs led to failures in clinical trials. More recent approaches have produced highly specific inhibitors to tackle selected MMPs, thus anticipating the development of more successful drugs in the near future. Further strategies should include the detailed structural characterization of the remaining ten MMPs to assist in achieving higher drug selectivity. In this review, we discuss the general architecture of MMP catalytic domains and its implication in function, zymogenic activation, and drug design. © 2009 Elsevier B.V.This study was supported by grants from Spanish ministries (BIO2006-02668, BIO2008-04080-E, BIO2009-10334, and CONSOLIDER-INGENIO 2010 Project “La Factoría de Cristalización” (CSD2006-00015)). Additional funding was obtained from the European Union through EU FP6 Strep Project LSHG-2006-018830 CAMP.Peer Reviewe

Structural and functional análisis of ulilysin, the prototype of the catalytic domain of the human IGFBP protease PAPP-A

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Molecular analysis of ulilysin, the structural prototype of a new family of metzincin metalloproteases

9 pages, 4 figures, 3 tables.-- PMID: [16627477].-- Printed version published Jun 30, 2006.This work was supported by Grant SAB2002-0102 from the Spanish Ministry for Education, Culture and Sports, Grant BIO2003-00132 from the Spanish Ministry for Science and Technology, Grant ON03-7-0 from the “Fundació La Caixa,” EU FP6 Integrated Project LSHC-CT-2003-503297, AVON Project 2005X0648 from the Spanish Association against Cancer, and Grants 31-67253.01 and 3100AO-108262/1 from the Swiss National Science Foundation. Funding for synchrotron diffraction data collection was provided by the European Synchrotron Radiation Facility (Grenoble, France).This work was supported by Grant SAB2002-0102 from the Spanish Ministry for Education, Culture and Sports, Grant BIO2003-00132 from the Spanish Ministry for Science and Technology, Grant ON03-7-0 from the “Fundació La Caixa,” EU FP6 Integrated Project LSHC-CT-2003-503297, AVON Project 2005X0648 from the Spanish Association against Cancer, and Grants 31-67253.01 and 3100AO-108262/1 from the Swiss National Science Foundation. Funding for synchrotron diffraction data collection was provided by the European Synchrotron Radiation Facility (Grenoble, France).Peer reviewe

Substrate specificity of a metalloprotease of the pappalysin family revealed by an inhibitor and a product complex

Human pappalysin-1 is a multi-domain metalloprotease engaged in the homeostasis of insulin-like growth factors and the founding member of the pappalysin family within the metzincin clan of metalloproteases. We have recently identified an archaeal relative, ulilysin, encompassing only the protease domain. It is a 262-residue active protease with a novel 3D structure with two subdomains separated by an active-site cleft. Despite negligible overall sequence similarity, noticeable similarity is found with other metzincin prototypes, adamalysins/ADAMs and matrix metalloproteinases. Ulilysin has been crystallised in a product complex with an arginine-valine dipeptide occupying the active-site S1′ and S2′ positions and in a complex with the broad-spectrum hydroxamic acid-based metalloprotease inhibitor, batimastat. This molecule inhibits mature ulilysin with an IC50 value of 61 μM under the conditions assayed. The binding of batimastat to ulilysin evokes binding to vertebrate matrix metalloproteases but is much weaker. These data give insight into substrate specificity and mechanism of action and inhibition of the novel pappalysin family. © 2006 Elsevier Inc. All rights reserved.This study was supported by the following grants and fellowships: SAB2002-0102 from the former Spanish Ministry for Education, Culture and Sports; BIO2003-00132, GEN2003-20642 and BIO2004-20369-E from the former Spanish Ministry for Science and Technology; BIO2006-02668 and BFU2006-09593 and CONSOLIDER-INGENIO 2010 Project ‘La Factorı´a de Cristalización’ (CSD2006-00015) from the Spanish Ministry for Education and Science; ON03-7-0 from the “Fundació La Caixa”; EU FP6 Integrated Project LSHC-CT-2003-503297 “CANCERDEGRADOME”; “AVON-Project” 2005X0648 from the Scientific Foundation of the Spanish Association Against Cancer; Grant 2005SGR00280 from the Generalitat of Catalunya; and grants 31-67253.01 and 3100AO-108262/1 from the Swiss National Science Foundation. M.S. is a beneficiary of the “Ramon y Cajal” Program from the Spanish Ministry for Science and EducationPeer Reviewe