scholarly journals Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state

2017 ◽  
Vol 15 (31) ◽  
pp. 6656-6667 ◽  
Author(s):  
Matthijs J. van Haren ◽  
Rebecca Taig ◽  
Jilles Kuppens ◽  
Javier Sastre Toraño ◽  
Ed E. Moret ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Substrate Binding ◽  
Structural Features ◽  
Methylation Reaction ◽  
Potent Inhibition ◽  
Binding Pockets

Inhibitors designed to simultaneously occupy the different substrate binding pockets of the NNMT active site reveal key structural features required for potent inhibition.

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets?

2003 ◽  
Vol 70 ◽  
pp. 15-30 ◽  
Author(s):  
Dŭsan Turk ◽  
Boris Turk ◽  
Vito Turk
Keyword(s):  
Drug Discovery ◽  
Active Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Cysteine Proteases ◽  
Structural Features ◽  
Discovery Research ◽  
Drug Discovery Research ◽  
Lysosomal Cysteine Proteases ◽  
Substrate Binding Sites

Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments will bring the desired result.

Structural and mechanistic insight into how antibodies inhibit serine proteases

2010 ◽  
Vol 430 (2) ◽  
pp. 179-189 ◽  
Author(s):  
Rajkumar Ganesan ◽  
Charles Eigenbrot ◽  
Daniel Kirchhofer
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Structural Changes ◽  
Competitive Inhibition ◽  
Substrate Binding ◽  
Structural Features ◽  
Inhibition Mechanism ◽  
Interaction Sites ◽  
Binding Cleft ◽  
Insight Into

Antibodies display great versatility in protein interactions and have become important therapeutic agents for a variety of human diseases. Their ability to discriminate between highly conserved sequences could be of great use for therapeutic approaches that target proteases, for which structural features are conserved among family members. Recent crystal structures of antibody–protease complexes provide exciting insight into the variety of ways antibodies can interfere with the catalytic machinery of serine proteases. The studies revealed the molecular details of two fundamental mechanisms by which antibodies inhibit catalysis of trypsin-like serine proteases, exemplified by hepatocyte growth factor activator and MT-SP1 (matriptase). Enzyme kinetics defines both mechanisms as competitive inhibition systems, yet, on the molecular level, they involve distinct structural elements of the active-site region. In the steric hindrance mechanism, the antibody binds to protruding surface loops and inserts one or two CDR (complementarity-determining region) loops into the enzyme's substrate-binding cleft, which results in obstruction of substrate access. In the allosteric inhibition mechanism the antibody binds outside the active site at the periphery of the substrate-binding cleft and, mediated through a conformational change of a surface loop, imposes structural changes at important substrate interaction sites resulting in impaired catalysis. At the centre of this allosteric mechanism is the 99-loop, which is sandwiched between the substrate and the antibody-binding sites and serves as a mobile conduit between these sites. These findings provide comprehensive structural and functional insight into the molecular versatility of antibodies for interfering with the catalytic machinery of proteases.

scholarly journals Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

2022 ◽  
Vol 12 ◽  
Author(s):  
Yeongjin Yun ◽  
Sangjun Han ◽  
Yoon Sik Park ◽  
Hyunjae Park ◽  
Dogyeong Kim ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Chemical Structure ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Zinc Ions ◽  
Substrate Binding Pocket ◽  
Inhibitory Spectrum

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

scholarly journals Norovirus Protease Structure and Antivirals Development

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2069
Author(s):  
Boyang Zhao ◽  
Liya Hu ◽  
Yongcheng Song ◽  
Ketki Patil ◽  
Sasirekha Ramani ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Active Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Conformational Flexibility ◽  
Current Status ◽  
Valuable Insight ◽  
Design And Development ◽  
Structural Differences ◽  
Binding Pockets

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is an excellent target for developing small-molecule inhibitors. The current strategy for developing HuNoV protease inhibitors is by targeting the enzyme’s active site and designing inhibitors that bind to the substrate-binding pockets located near the active site. However, subtle differential conformational flexibility in response to the different substrates in the polyprotein and structural differences in the active site and substrate-binding pockets across different genogroups, hamper the development of effective broad-spectrum inhibitors. A comparative analysis of the available HuNoV protease structures may provide valuable insight for identifying novel strategies for the design and development of such inhibitors. The goal of this review is to provide such analysis together with an overview of the current status of the design and development of HuNoV protease inhibitors.

scholarly journals Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin

1988 ◽  
Vol 256 (2) ◽  
pp. 357-361 ◽  
Author(s):  
P Hadváry ◽  
H Lengsfeld ◽  
H Wolfer
Keyword(s):  
Transition State ◽  
Acid Derivative ◽  
Pancreatic Lipase ◽  
Active Site ◽  
Boronic Acid ◽  
Selective Inhibitor ◽  
Covalent Intermediate ◽  
State Form ◽  
Covalent Inhibitor

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.

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