ABSTRACT
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Subject(s)
Benzamidines/chemistry , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , Serine Proteinase Inhibitors/chemistry , Amino Acid Sequence , Benzamidines/pharmacology , Binding Sites , Drug Evaluation, Preclinical , Humans , Isomerism , Models, Molecular , Molecular Structure , Mutation , Protein Binding , Serine Peptidase Inhibitor Kazal-Type 5/genetics , Serine Proteinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.
Subject(s)
Benzamidines/chemistry , Kallikreins/chemistry , Protease Inhibitors/chemistry , Amino Acid Sequence , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Benzamidines/pharmacology , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Drug Discovery , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kallikreins/antagonists & inhibitors , Kallikreins/genetics , Kallikreins/metabolism , Kinetics , Models, Molecular , Mutation , Netherton Syndrome/drug therapy , Netherton Syndrome/enzymology , Protease Inhibitors/pharmacology , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , Spodoptera , Static Electricity , Substrate SpecificityABSTRACT
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.
Subject(s)
Drug Design , Kallikreins/antagonists & inhibitors , Netherton Syndrome/drug therapy , HumansABSTRACT
Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.
Subject(s)
Benzamidines/chemistry , Kallikreins/antagonists & inhibitors , Serine Proteinase Inhibitors/chemistry , Animals , Benzamidines/chemical synthesis , Benzamidines/metabolism , Catalytic Domain , Drug Design , Kallikreins/metabolism , Netherton Syndrome/drug therapy , Protein Binding , Salicylamides/chemical synthesis , Salicylamides/chemistry , Salicylamides/metabolism , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/metabolism , Spodoptera/geneticsABSTRACT
The pharmacological properties of the novel ligand, (2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol (I), at the human adenosine receptors were investigated using Chinese hamster ovary cell lines recombinantly expressing these receptors. Functional studies were performed using a cyclic AMP-coupled reporter gene system. Binding studies were performed using membranes from these cells. The effects of ligand (I) were also determined on functional responses of human neutrophils and eosinophils. Ligand (I) had a high affinity for the adenosine A(2A) receptor (pKi 7.8+/-0.2) and was a potent agonist at this receptor (pEC(50) 9.0+/-0.2). Ligand (I) had a similar affinity for the adenosine A(3) receptor (pKi 7.8+/-0.1) but displayed no agonist activity, acting instead as a competitive antagonist (pA(2) 8.3+/-0.04). Ligand (I) had lower affinity for adenosine A(1) and A(2B) receptors (pKi
