Insights into the activity control of the kallikrein-related peptidase 6: small-molecule modulators and allosterism (original) (raw)
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Chemistry & Biology, 2009
Human kallikrein-related peptidase 4 (hk4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment as a consequence of its proteolytic ability to activate many tumorigenic and metastatic pathways such as the protease activated receptors (PARs). Currently there are no KLK4specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally-occurring sunflower trypsin inhibitor (SFTI) to specifically block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in cell based systems. The re-engineered inhibitor was designed using a combination of molecular modelling and sparse matrix substrate screening. The resulting inhibitor showed highly specific inhibition of KLK4 against the peptide substrate FVQR-para-nitroanilide (K i of 3.25 ± 1.60 nM) and the macromolecular substrate fibrinogen, blocked KLK4 initiated calcium flux from PAR2 and displayed remarkable stability in cell based assays with a half life of four days.
The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding His57 undergoes an outward rotation thus the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by covalent attachment of aromatic coumarinic esters.
Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 and 34 have single digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivity, respectively, against the closely related enzyme trypsin. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validatio...
Biological chemistry, 2016
Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesised peptide substrates to characterise the binding specificity of KLK5. The sequences most favoured by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analysing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki = 4.2 ± 0.2 nM), weak activity against KLK7 an...
Inhibitors of kallikrein‐related peptidases: An overview
Medicinal Research Reviews, 2017
Kallikrein‐related peptidases (KLKs) are a family of 15 secreted serine proteases that are involved in various physiological processes. Their activities are subtly regulated by various endogenous inhibitors, ranging from metallic ions to macromolecular entities such as proteins. Furthermore, dysregulation of KLK activity has been linked to several pathologies, including cancer and skin and inflammatory diseases, explaining the numerous efforts to develop KLK‐specific pharmacological inhibitors as potential therapeutic agents. In this review, we focus on the huge repertoire of KLKs inhibitors reported to date with a special emphasis on the diversity of their molecular mechanisms of inhibition.
Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs)
Biochimie, 2010
Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn 2þ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming a 2 -macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.
Defining the extended substrate specificity of kallikrein 1-related peptidases
bchm, 2007
Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1–P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2–P4 specificities, attributable to structural variations in their surface loops. The preferred P4–P1 substrate recognition motifs based on optimal subsite occupancy were as follows: ...
Biopolymers, 2013
Potent and specific enzyme inhibition is a key goal in development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclised peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry.
The solution structure of the kallikrein-related peptidases inhibitor SPINK6
Biochemical and biophysical research communications, 2016
Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.
Natural and engineered kallikrein inhibitors: an emerging pharmacopoeia
Biological Chemistry, 2000
The kallikreins and kallikrein-related peptidases are serine proteases which control a plethora of developmental and homeostatic phenomena ranging from semen liquefaction to skin desquamation and blood pressure. The diversity of roles played by kallikreins has stimulated considerable interest in these enzymes from the perspective of diagnostics and drug design.