RESUMO
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.
Assuntos
Desenho de Fármacos , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , HumanosRESUMO
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.
Assuntos
Benzamidinas/química , Calicreínas/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Animais , Benzamidinas/síntese química , Benzamidinas/metabolismo , Domínio Catalítico , Desenho de Fármacos , Calicreínas/metabolismo , Síndrome de Netherton/tratamento farmacológico , Ligação Proteica , Salicilamidas/síntese química , Salicilamidas/química , Salicilamidas/metabolismo , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/metabolismo , Spodoptera/genéticaRESUMO
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.
Assuntos
Benzamidinas/química , Calicreínas/antagonistas & inibidores , Síndrome de Netherton/tratamento farmacológico , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Benzamidinas/farmacologia , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Humanos , Isomerismo , Modelos Moleculares , Estrutura Molecular , Mutação , Ligação Proteica , Inibidor de Serinopeptidase do Tipo Kazal 5/genética , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Benzamidinas/química , Calicreínas/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Descoberta de Drogas , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/metabolismo , Cinética , Modelos Moleculares , Mutação , Síndrome de Netherton/tratamento farmacológico , Síndrome de Netherton/enzimologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática , Especificidade por SubstratoRESUMO
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=6) and showed relatively weak agonist activity at these receptors (pEC(50) 7.1 at both receptors). Ligand (I) was a potent inhibitor of the generation of reactive oxygen species from human neutrophils and eosinophils (pEC(50) 9.7+/-0.1 and 9.4+/-0.2 respectively). The inhibitory effect of ligand (I) on the release of reactive oxygen species from neutrophils was antagonised competitively by the adenosine A(2A) receptor antagonist 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) with a pA(2) value (10.03+/-0.44) consistent with an effect on adenosine A(2A) receptors. Ligand (I) also inhibited the release of granule proteins from neutrophils and eosinophils (pEC(50) 8.7 and 8.9 respectively), albeit less potently than as an inhibitor of reactive oxygen species generation. In summary, ligand (I) is a potent and selective agonist for the adenosine A(2A) receptor and a competitive antagonist at the adenosine A(3) receptor. Ligand (I) has potent anti-inflammatory effects on human granulocytes in vitro.