Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Kenniston, Jon A.; Faucette, Ryan; Martik, Diana; Comeau, Stephen R.; Lindberg, Allison P.; Kopacz, Kris; Conley, Gregory P.; Chen, Jie; Viswanathan, Malini; Kastrapeli, Niksa; Cosic, Janja; Mason, Shauna; DiLeo, Mike; Abendroth, Jan; Kuzmič, Petr; Ladner, Robert C.; Edwards, Thomas E.; TenHoor, Christopher; Adelman, Burt; Nixon, Andrew E.; Sexton, Daniel J.

doi:10.1074/jbc.m114.569061

Cited by 100 publications

(122 citation statements)

References 74 publications

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“…Moreover, the P1 Arg residue forms a suboptimal interaction in which a water molecule is required to mediate the interaction between the P1 Arg residue and Asp-189 at the bottom of the S1 pocket. In contrast, the anti-plasma kallikrein antibody DX-2930 binds in the normal N-to C-terminal orientation, but a non-substrate-like conformation of the loop prevents it from binding the S1Ј-S3Ј pockets and thereby hydrolysis of the putative scissile bond (21). The anti-HGFA Fab58 also binds to the active site region of the protease, occluding the S2 and S3 but not the S1 pocket (20).…”

Section: Discussionmentioning

confidence: 99%

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Kromann-Hansen

Oldenburg

Yung

et al. 2016

Journal of Biological Chemistry

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A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30 -40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.Serine proteases catalyze the hydrolysis of peptide bonds and are involved in numerous physiological processes, including digestion, blood clotting, fibrinolysis, complement activation, and turnover of the extracellular matrix (1). Neutralizing serine protease activity using orthosteric inhibitors, i.e. active site binding inhibitors, has been shown to be a successful therapeutic strategy for a number of pathological conditions, although the similar active site topology in all serine proteases increases the risk of off-target effects. Today, serine protease inhibitors are clinically used for therapy of several diseases, including thrombosis and bleeding disorders (2-4).All serine proteases catalyze the same type of hydrolytic reaction utilizing the same biochemical mechanism. Serine protease-catalyzed hydrolysis of a scissile bond proceeds through a highly conserved mechanism involving two tetrahedral intermediates and an acyl-enzyme complex. The polypeptide substrate is aligned in the active site of the protease interacting with the substrate specificity pockets denoted S1-Sn and S1Ј-SnЈ on the acyl and leaving group side of the scissile bond, respectively (5). The P1 residue of the substrate binds into the S1 pocket, and its carbonyl oxygen atom is inserted into the so-called oxyanion hole (backbone amides of chymotrypsinogen numbering). The catalytic triad (His-57, in the protease generates the required nucleophile for the attack of the hydroxyl group of Ser-195 on the carbonyl group of the P1-P1Ј scissile bond to form the first tetrahedral intermediate and later the acyl-enzyme. Following release of the P1Ј-leaving group, a water molecule performs a second nucleophilic attack, thereby completing the cycle (6).Peptide segments that bind the active site of serine proteases in a substrate-like manner may behave like an inhibitor or substrate. Ho...

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Section: Discussionmentioning

confidence: 99%

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Kromann-Hansen

Oldenburg

Yung

et al. 2016

Journal of Biological Chemistry

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“…1).415 To complicate matters further, our own experience shows that 416 the randomness in the distribution of residuals, when considered 417 in isolation, is not a suitable measure of significant nonlinearity 418 in enzyme kinetics. If fact, we found that in certain exquisitely419 well-tuned enzyme assays[19], the random scatter in the data 420 points is so low that even insignificant departures from linearity 421 (e.g., those observed at extremely low final substrate conversions)422 will produce decidedly nonrandom residual patterns.423 Based on similar practical reasons, we hereby propose an oper-424 ational definition of nonlinearity in the context of covalent enzyme425 inhibition kinetics, which is inspired by the ''cross-validation'' con-426 cept from statistics[16]. The basic idea is to divide the control pro-427 gress curve into two halves, fit each segment to the straight line 428 model, and finally compare the two resulting slopes, that is, the 429 ''initial'' and ''final'' reaction rates.…”

mentioning

confidence: 97%

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

Kuzmič¹,

Solowiej

Murray

2015

Analytical Biochemistry

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“…A second phase is mediated by bradykinin. An antibody blocking PKal, an enzyme that cleaves high molecular weight kininogen to generate bradykinin, produced significant inhibition of edema in this model [249].…”

Section: Surrogate Non-ocular Modelsmentioning

confidence: 99%

Selection Strategy of In Vivo Models for Ophthalmic Drug Development in Diabetic Retinopathy

Geert¹,

Tjing-Tjing²

2016

J Mol Genet Med

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Diabetic Retinopathy (DR) is the most common microvascular complication of diabetes and is one of the leading causes of visual impairment worldwide. DR is a chronic eye disease that eventually can result in legal blindness due to the evolution towards the major vision-threatening disorders diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR). Current treatments with steroids, anti-VEGF compounds or retinal laser photocoagulation have shown a significant improvement in visual acuity in the advanced stage of the disease. However, main concerns are possible side effects and/or the relatively large number of clinical non-responders. A better understanding of the biological and molecular pathways not only in DR patients but also in the preclinical in vivo models would aid the development of novel and more efficient (personalized) therapeutic approaches for DR. This review aims to describe the pathways in a selection of diabetic, non-diabetic and surrogate rodent models of pathogenic neovascularization, vascular permeability, inflammation and neurodegeneration. None of these models can mimic the entire human pathophysiological progression but they all exhibit joint pathophysiological features with human DR pathogenesis. These combined features make these models very relevant in gaining a better understanding of the disease etiology and eventually improved strategies for drug screening. Through highlighting the most important biochemical and molecular pathways that these animal models have in common with DR patients, selection of the most suitable model for mechanistic studies and drug screening can be facilitated.

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Inhibition of Plasma Kallikrein by a Highly Specific Active Site Blocking Antibody

Cited by 100 publications

References 74 publications

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

Selection Strategy of In Vivo Models for Ophthalmic Drug Development in Diabetic Retinopathy

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