RESUMO
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 the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.
Assuntos
Cumarínicos/química , Calicreínas/antagonistas & inibidores , Inibidores de Proteases/química , Sítios de Ligação , Domínio Catalítico , Cumarínicos/metabolismo , Cristalografia por Raios X , Ésteres/química , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure-activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,ß-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo- and 2-chloro-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (15, 16) being the most potent inhibitors with Ki values toward human CD73 of 3-6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP (21) was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky N6-substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Difosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Animais , Cristalografia por Raios X , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,ß-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Difosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/química , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Ratos , SpodopteraRESUMO
In vertebrates ecto-5'-nucleotidase (e5NT) catalyzes the hydrolysis of extracellular AMP to adenosine and represents the major control point for extracellular adenosine levels. Due to its pivotal role for activation of P1 adenosine receptors, e5NT has emerged as an appealing drug target for treatment of inflammation, chronic pain, hypoxia, and cancer. Crystal structures of the dimeric human e5NT reveal an extensive 114° conformational switch between the open and closed forms of the enzyme. The dimerization interface is formed by the C-terminal domains and exhibits interchain motions of up to 13°. Complex structures with adenosine and AMPCP indicate that structural control of the domain movement determines the selectivity for monophosphate nucleotides. Binding modes of nucleotide-derived and flavonoid-based compounds complexed to the C-terminal domain in the open form reveal an additional binding pocket of â¼210 Å(3) that might be explored to design more potent inhibitors.