Characterization ofP. falciparumdipeptidyl aminopeptidase 3 specificity reveals structural factors responsible for differences in amino acid preferences between peptide-based substrates and covalent inhibitors

Abstract

Dipeptidyl aminopeptidases (DPAPs) are druggable cysteine proteases that cleave dipeptides from the N-terminus of proteins and oligopeptides. Plasmodium DPAPs have been shown to be important for the asexual replication of the malaria parasite and are therefore potential antimalarial targets. DPAP1 seems to be important for parasite growth within infected red blood cells, and DPAP3 plays a critical role in the invasion of erythrocytes by the malaria parasite. An inhibitor able to block both of these proteases will target the parasite at different developmental stages thus making the emergence of drug resistance more difficult.Understanding the substrate specificity of these proteases is important to understand their molecular functions and to help develop potent and selective inhibitors. In this study, we used peptide-based libraries of substrates and inhibitors to determine the specificity of DPAP3 and compared it to that of DPAP1 and human cathepsin C (mammalian DPAP homologue). We then used the structure activity relationships information from these screens to develop optimal DPAP3 fluorogenic substrates as well as highly specific DPAP1 and DPAP3 inhibitors.Interestingly, while the substrate specificity of a protease is often used to develop potent inhibitors, in this study we show that equally potent and highly specific inhibitors can be developed based on the structure of poor substrates. Overall, this study illustrates that focusing the development of peptidic inhibitors solely on the substrate specificity of a protease might overlook important structural features that can be exploited to developed highly potent and selective compounds.not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/246124 doi: bioRxiv preprint first posted online Jan. 11, 2018; 3 Malaria is a devastating infectious parasitic disease causing close to half a million deaths every year 1 . Over the last 15 years the world has seen a very significant drop in malaria incidence, mainly due to the global distribution of insecticide-impregnated bed nets and the use of artemisinin-based combination therapies as the standard of care for uncomplicated malaria 2 .However, malaria remains a major global health burden with half of the world population at risk and around 200 million clinical cases per year. Unfortunately, mosquitoes are becoming increasingly resistance to insecticides 3 and artemisinin resistance is on the rise 4 , thus making the identification of antimalarial targets and the development of drugs with novel mechanisms of action extremely urgent 5 .Malaria is caused by parasites of the Plasmodium genus and is transmitted by Anopheles mosquitoes during a blood meal. Upon infection, malaria parasites first establish an asymptomatic infection in the liver before reaching the blood stream where they replicate asexually through multiple rounds of red blood cell (RBC) invasion, intracellular growth and divis...

Identification of Plasmodium dipeptidyl aminopeptidase allosteric inhibitors by high throughput screening

“…The synthesis and characterization of the fluorogenic substrates (PR) 2 Rho[28], VR-ACC[29], and nVal-nLeu(o-Bzl)-ACC[30] have been previously described. Z-LR-AMC and GR-AMC were purchased from Sigma.…”

“…Each protease was used at 1 nM in the HTS assay buffer described above. Dose response inhibition studies to obtained IC 50 values for each of the proteases tested were performed using fluorogenic substrates at the K m concentrations determined under our assay conditions: VR-ACC for DPAP1 ( K m,DPAP1 = 20 μM)[29], nVal-nLeu(o-Bzl)-ACC for DPAP3 ( K m,DPAP3 = 1.4 μM)[30], GR-AMC for CatC ( K m,CatC = 40 μM), and Z-LR-AMC for the FPs ( K m,FP2 = 5 μM; K m,FP3 = 20 μM). For all assays, substrate turnover was measured for 30 min at 460 nm (λ ex = 355nm) using a SpectraMax M5e (Molecular Devices) multimode plate reader.…”

“…CatC (2 μM) was incubated for 30 min in assay buffer with DMSO or 20 μM of JCP410, a very potent covalent inhibitor of CatC ( k i / K i = 5.8x10 6 M -1 s -1 )[30]. The enzyme was then diluted 2-fold in assay buffer containing different concentrations of inhibitors.…”