Proteases as therapeutics

Craik, Charles S.; Page, Michael; Madison, Edwin L.

doi:10.1042/bj20100965

Cited by 204 publications

(173 citation statements)

References 186 publications

(177 reference statements)

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“…Thus, transmutation of only four SDP residues changed the proteolytic function of MMP-16 into that of MMP-17. We believe that this and similar knowledge may contribute to the redesign of MMPs to exhibit unconventional substrate specificity, which could have therapeutic, diagnostic, or other biotechnology applications (56,57). The substrate specificity of several proteases distinct from MMPs has been manipulated (58)(59)(60), but this was thought to be difficult to achieve with MMPs because of their presumed overlapping substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

Basis for substrate recognition and distinction by matrix metalloproteinases

Ratnikov¹,

Cieplak²,

Gramatikoff³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for functionrelated predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k (obs) ] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.protease | specificity-determining positions | MMPs A paramount objective of biological research is to understand how sequence encodes function. Previously, functional regions in proteins were identified using large-scale mutagenesis (e.g., alanine scanning) (1). More recently, our insights are largely gained by computational approaches aimed at comparing sequences and structures of large protein sets across multiple genomes. The vast increase in the number of available sequences makes it possible to compare homology between sequences from genome projects to proteins of known structure and function and, as a result, identify functional similarities in silico (2-4). Because the global fold of most ordered proteins can be reliably predicted (5, 6) and because the catalytic residues of most classes of enzymes are either known or can be inferred (7,8), protein sequences can now be directly used to elucidate and classify major protein functions (9-12), and are even being extended to predict enzyme substrates (13).However, such classifications fail to explain the specialization and expansion of function that is required for organismal plasticity, complexity, and adaptability, all of which are normally driven by gene duplications and subsequent divergence. Computational approaches aimed at identifying functional distinctions across protein families have pri...

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Section: Discussionmentioning

confidence: 99%

Basis for substrate recognition and distinction by matrix metalloproteinases

Ratnikov¹,

Cieplak²,

Gramatikoff³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The molecular basis of autoactivation in terms of conformational selection also defines a strategy for the facile production of trypsin-like proteases of clinical and biotechnological relevance. Tissue-type plasminogen activator, thrombin, and activated protein C are or have been on the market for the treatment of medically relevant conditions such as stroke, hemorragic complications, and sepsis (46). Thrombin variants engineered for anticoagulant and antithrombotic activity in vivo are in preclinical stage (47).…”

Section: Discussionmentioning

confidence: 99%

Autoactivation of Thrombin Precursors

Pozzi

Chen

Zapata

et al. 2013

Journal of Biological Chemistry

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Background: Thrombin is generated from zymogen precursors with the assistance of cofactors. Results: Thrombin precursors prethrombin-2 and prothrombin are capable of catalytic activity and autoactivate. Conclusion: Conformational selection regulates activity in the mature protease and has the potential to unleash autoactivation in the zymogen. Significance: The paradigm of the inactive zymogen to active protease conversion needs revision to account for conformational selection.

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“…They are related in almost every aspects of life such as protein turnover, sporulation, germination, enzyme modification, blood clotting, tumor progression etc. 1 Their industrial applications include detergents, food industry, leather industry, manufacture of aspartame etc. 2 Instead of complicated protein biology, proteases are relatively safe and well tolerated which enables their applications in thrombolysis, correction of coagulation disorders, sepsis, neuromascular problems, digestion, etc.…”

Section: Introductionmentioning

confidence: 99%

“…2 Instead of complicated protein biology, proteases are relatively safe and well tolerated which enables their applications in thrombolysis, correction of coagulation disorders, sepsis, neuromascular problems, digestion, etc. 1 Formation of clots (thrombus) in blood vessels can lead to serious disease to human such as myocardial infarction and stroke. Drugs using fibrinolytic enzymes are the most effective methods in the treatment of thrombosis.…”

Section: Introductionmentioning

confidence: 99%

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Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

Zaman

Mamun

Khan

et al. 2017

Dhaka Univ. J. Pharm. Sci

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Thrombosis leads to myocardial infarction, stroke and other cardiovascular complications. Microbial thrombolytic agents such as urokinase, streptokinase etc. are used to treat complications related to thrombosis. To search for new microbial enzymes as thrombolytics having better efficacy and specificity, Bacillus licheniformis EMS-O-1 mutant strain was cultured in modified urea-molasses media followed by purification using ammonium sulphate precipitation and ultrafiltration through centricon tube of 100 MWCO value. The yield of crude enzyme was 11129.14 U/mg and after purification 40180.46 U/mg. Purification process increased the specific activity of purified enzyme to 12.28 fold with a recovery of 17.79%. The purified enzyme was a serine protease with molecular weight of 25.5 kDa as confirmed by irreversible inhibition of activity with phenylmethylsulfonyl fluoride (PMSF) followed by SDS-PAGE gel image and by LC-MS analyses. In vitro clot lysis assay of the purified enzyme exhibited 38.30% thrombolytic activity. The crude enzymes from the mutant strain EMS-O-1 were found to be stable up to 50 o C and showed maximum stability between pH range 7.5 to 8.5. These findings signify that proteases produced by B. licheniformis mutant have the potential to be developed as a viable thrombolytic agent.

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Proteases as therapeutics

Cited by 204 publications

References 186 publications

Basis for substrate recognition and distinction by matrix metalloproteinases

Basis for substrate recognition and distinction by matrix metalloproteinases

Autoactivation of Thrombin Precursors

Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

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