RESUMO
Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.
Assuntos
Alanina/metabolismo , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Endopeptidases/metabolismo , Mutação , Serina Endopeptidases/metabolismo , Alanina/química , Alanina/genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Sítios de Ligação/genética , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Linhagem Celular , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases/genética , Gelatinases , Humanos , Proteínas de Membrana , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Relação Estrutura-Atividade , Especificidade por Substrato/genéticaRESUMO
The structure-activity relationship of various N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro derivatives against three prolyl peptidases was explored. Several N-acyl-Gly- and N-blocked-boroPro compounds showed low nanomolar inhibitory activity against fibroblast activation protein (FAP) and prolyl oligopeptidase (POP) and selectivity against dipeptidyl peptidase-4 (DPP4). N-Acyl-Sar-boroPro analogs retained selectivity against DPP4 and potent POP inhibitory activity but displayed decreased FAP inhibitory activity.
Assuntos
Compostos de Boro/síntese química , Prolina/química , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Inibidores de Adenosina Desaminase , Antígenos de Neoplasias , Biomarcadores Tumorais/antagonistas & inibidores , Compostos de Boro/farmacologia , Dipeptidil Peptidase 4 , Inibidores da Dipeptidil Peptidase IV , Endopeptidases , Gelatinases , Glicoproteínas/antagonistas & inibidores , Proteínas de Membrana , Prolina/farmacologia , Prolil Oligopeptidases , Serina Endopeptidases/efeitos dos fármacos , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.
Assuntos
Antígenos de Neoplasias/química , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Desenho de Fármacos , Serina Endopeptidases/química , Serina Endopeptidases/efeitos dos fármacos , Inibidores de Serina Proteinase/química , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/farmacologia , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/efeitos dos fármacos , Endopeptidases , Gelatinases , Humanos , Proteínas de Membrana , Modelos Moleculares , Oligopeptídeos/química , Peptídeos/química , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , alfa 2-Antiplasmina/químicaRESUMO
Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.