Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening

2019 ◽  
Vol 476 (3) ◽  
pp. 499-512 ◽  
Author(s):  
Simon Law ◽  
Xin Du ◽  
Preety Panwar ◽  
Nicolette S. Honson ◽  
Tom Pfeifer ◽  
...  
Keyword(s):  
Active Site ◽  
High Throughput Screening ◽  
Drug Target ◽  
Cathepsin K ◽  
Peptide Substrate ◽  
Collagenase Activity ◽  
Elastase Activity ◽  
Specific Selectivity ◽  
Proteolytic Activities ◽  
Hydrolysis Of

Abstract Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Structural requirements for the collagenase and elastase activity of cathepsin K and its selective inhibition by an exosite inhibitor

2014 ◽  
Vol 465 (1) ◽  
pp. 163-173 ◽  
Author(s):  
Vidhu Sharma ◽  
Preety Panwar ◽  
Anthony J. O’Donoghue ◽  
Haoran Cui ◽  
Rafael V. C. Guido ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Extracellular Matrix Proteins ◽  
Cathepsin K ◽  
Selective Inhibition ◽  
Matrix Proteins ◽  
Elastase Activity ◽  
Structural Requirements ◽  
Proteolytic Activities

The degradation of extracellular matrix proteins such as elastin and collagen by cathepsin K is selectively regulated by exosites. A specific exosite inhibitor blocks the collagenase and elastase activity without interfering with other proteolytic activities.

Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site

Biochemistry ◽  
1990 ◽  
Vol 29 (28) ◽  
pp. 6670-6677 ◽  
Author(s):  
Gregg B. Fields ◽  
Sarah J. Netzel-Arnett ◽  
Lester J. Windsor ◽  
Jeffrey A. Engler ◽  
Henning Birkedal-Hansen ◽  
...  
Keyword(s):  
Active Site ◽  
Human Fibroblast ◽  
Fibroblast Collagenase ◽  
Proteolytic Activities ◽  
Hydrolysis Of

scholarly journals The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

2020 ◽  
Vol 59 (51) ◽  
pp. 22916-22921
Author(s):  
Himanshu Singh ◽  
Chandan K. Das ◽  
Suresh K. Vasa ◽  
Kristof Grohe ◽  
Lars V. Schäfer ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Conformational Dynamics

scholarly journals A Mechanism-Based Whole-Cell Screening Assay to Identify Inhibitors of Protein Export in Escherichia coli by the Sec Pathway

2012 ◽  
Vol 17 (4) ◽  
pp. 535-541 ◽  
Author(s):  
Gregory J. Crowther ◽  
S. Arshiya Quadri ◽  
Benjamin J. Shannon-Alferes ◽  
Wesley C. Van Voorhis ◽  
Henry Rosen
Keyword(s):  
Escherichia Coli ◽  
High Throughput Screening ◽  
Drug Target ◽  
Rapid Identification ◽  
Protein Synthesis Inhibitors ◽  
Screening Assay ◽  
Whole Cell ◽  
Sec Pathway ◽  
Associated Proteins ◽  
Pass Through

More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (β-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent β-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay’s readiness for larger screening projects.

The 11S proteasome activator: Isolation from mouse brain and the influence on peptide substrate hydrolysis of the 20S and 26S proteasomes

2016 ◽  
Vol 71 (2) ◽  
pp. 97-103 ◽  
Author(s):  
A. V. Bacheva ◽  
O. V. Korobkina ◽  
P. S. Nesterova ◽  
V. A. Kryachkov ◽  
A. G. Gabibov
Keyword(s):  
Mouse Brain ◽  
Peptide Substrate ◽  
Proteasome Activator ◽  
Hydrolysis Of ◽  
Substrate Hydrolysis

scholarly journals The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor

2016 ◽  
Vol 72 (11) ◽  
pp. 813-819 ◽  
Author(s):  
Tony Christopeit ◽  
Ke-Wu Yang ◽  
Shao-Kang Yang ◽  
Hanna-Kirsti S. Leiros
Keyword(s):  
Public Health ◽  
Crystal Structure ◽  
Active Site ◽  
Drug Target ◽  
Zinc Ions ◽  
Clinical Use ◽  
Global Public Health ◽  
Promising Strategy ◽  
Conserved Residue ◽  
Substrate Spectrum

The increasing number of pathogens expressing metallo-β-lactamases (MBLs), and in this way achieving resistance to β-lactam antibiotics, is a significant threat to global public health. A promising strategy to treat such resistant pathogens is the co-administration of MBL inhibitors together with β-lactam antibiotics. However, an MBL inhibitor suitable for clinical use has not yet been identified. Verona integron-encoded metallo-β-lactamase 2 (VIM-2) is a widespread MBL with a broad substrate spectrum and hence is an interesting drug target for the treatment of β-lactam-resistant infections. In this study, three triazolylthioacetamides were tested as inhibitors of VIM-2. One of the tested compounds showed clear inhibition of VIM-2, with an IC50of 20 µM. The crystal structure of the inhibitor in complex with VIM-2 was obtained by DMSO-free co-crystallization and was solved at a resolution of 1.50 Å. To our knowledge, this is the first structure of a triazolylthioacetamide inhibitor in complex with an MBL. Analysis of the structure shows that the inhibitor binds to the two zinc ions in the active site of VIM-2 and revealed detailed information on the interactions involved. Furthermore, the crystal structure showed that binding of the inhibitor induced a conformational change of the conserved residue Trp87.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

scholarly journals Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase

1992 ◽  
Vol 286 (1) ◽  
pp. 23-30 ◽  
Author(s):  
M F Hoylaerts ◽  
T Manes ◽  
J L Millán
Keyword(s):  
Germ Cell ◽  
Active Site ◽  
Molecular Model ◽  
Inhibition Mechanism ◽  
Side Group ◽  
Alkaline Phosphatases ◽  
Uncompetitive Inhibition ◽  
Guanidinium Group ◽  
Hydrolysis Of ◽  
Site Binding

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

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