RESUMO
p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.
Assuntos
Permeabilidade da Membrana Celular , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fenilalanina/síntese química , Piridonas/síntese química , Domínios de Homologia de src , Células CACO-2 , Cálcio/metabolismo , Humanos , Células Jurkat , Ligantes , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Piridonas/química , Piridonas/farmacologiaRESUMO
Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.
Assuntos
Inibidores da Protease de HIV/síntese química , Piridinas/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacocinética , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Ligantes , Modelos Moleculares , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.
Assuntos
Dipeptídeos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Domínios de Homologia de src , Cristalografia por Raios X , Dipeptídeos/química , Ligantes , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.
Assuntos
Dipeptídeos/síntese química , Inibidores Enzimáticos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosfotirosina/química , Domínios de Homologia de src , Ligação Competitiva , Dipeptídeos/química , Dipeptídeos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has been synthesized. These peptidomimetic structures inhibit viral protease activity at low nanomolar concentrations (IC50 < 10 nM for HIV-1 protease). The inhibition constant (Ki) for inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases, respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays with 50% effective concentrations (EC50) = 3.7-35 nM. This series of inhibitors was found to exhibit poor bioavailability (< 10%) in the rat, following oral administration. The synthesis and biological properties of these compounds are discussed. In addition, an X-ray structure of one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding mode of this novel class of HIV protease inhibitors.