Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-013-1654-2) contains supplementary material, which is available to authorized users.
Seven crystal structures of alanyl
aminopeptidase from Neisseria meningitides (the etiological
agent of meningitis, NmAPN) complexed with organophosphorus
compounds were resolved
to determine the optimal inhibitor–enzyme interactions. The
enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond
to the P1 amino acid residues of well-processed substrates, were used
to assess the impact of the absolute configuration and the stereospecific
hydrogen bond network formed between the aminophosphonate polar head
and the active site residues on the binding affinity. For the hPhe
analog, an imperfect stereochemical complementarity could be overcome
by incorporating an appropriate P1 side chain. The constitution of
P1′-extended structures was rationally designed and the lead,
phosphinic dipeptide hPhePψ[CH2]Phe, was modified
in a single position. Introducing a heteroatom/heteroatom-based fragment
to either the P1 or P1′ residue required new synthetic pathways.
The compounds in the refined structure were low nanomolar and subnanomolar
inhibitors of N. meningitides, porcine and human
APNs, and the reference leucine aminopeptidase (LAP). The unnatural
phosphinic dipeptide analogs exhibited a high affinity for monozinc
APNs associated with a reasonable selectivity versus dizinc LAP. Another
set of crystal structures containing the NmAPN dipeptide
ligand were used to verify and to confirm the predicted binding modes;
furthermore, novel contacts, which were promising for inhibitor development,
were identified, including a π–π stacking interaction
between a pyridine ring and Tyr372.
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