Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity

Background: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery.Methodology/Principal findings: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 &lt; 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of &gt;500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.Conclusions/Significance: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.</p

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans

Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans.This research was supported under the Fellowship for short stays to prestigious researchers awarded by the University of Cadiz (reference EST2019-021).Peer reviewe

Insights into the Adsorption Mechanisms of the Antimicrobial Peptide CIDEM-501 on Membrane Models

CIDEM-501 is a hybrid antimicrobial peptide rationally designed based on the structure of panusin and panulirin template peptides. The new peptide exhibits significant antibacterial activity against multidrug-resistant pathogens (MIC = 2–4 μM) while conserving no toxicity in human cell lines. We conducted molecular dynamics (MD) simulations using the CHARMM-36 force field to explore the CIDEM-501 adsorption mechanism with different membrane compositions. Several parameters that characterize these interactions were analyzed to elucidate individual residues’ structural and thermodynamic contributions. The membrane models were constructed using CHARMM-GUI, mimicking the bacterial and eukaryotic phospholipid compositions. Molecular dynamics simulations were conducted over 500 ns, showing rapid and highly stable peptide adsorption to bacterial lipids components rather than the zwitterionic eucaryotic model membrane. A predominant peptide orientation was observed in all models dominated by an electric dipole. The peptide remained parallel to the membrane surface with the center loop oriented to the lipids. Our findings shed light on the antibacterial activity of CIDEM-501 on bacterial membranes and yield insights valuable for designing potent antimicrobial peptides targeting multi- and extreme drug-resistant bacteria

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BackgroundChagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery.Methodology/principal findingsA RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.Conclusions/significanceOur data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease

Potency determination for LAPTc screening hits.

Compound structures and pIC50 values against LAPTc as determined by dose-response studies with the RapidFire-MS enzymatic assay. Data from four independent replicates. * = one replicate was deemed inactive (maximum effect < 50%), average and standard deviation are for the three active replicates.</p

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.</p

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].</p

LAPTc pIC50 DRC.xlsx: Potency data for hits in RapidFire-MS assay. pIC₅₀ = -LOG(IC₅₀[M]).

Experiment column indicates if potency was obtained from initial potency confirmation or subsequent replicate generation experiments. Hit confirmation column indicates if hits from primary single concentration screen confirmed in the potency experiment. (XLSX)</p

Determination of the mode of inhibition of LAPTc by compound 4, with respect to the LSTVIVR peptide substrate using the RapidFire-MS enzymatic assay.

Double reciprocal Lineweaver-Burk plots are shown. Determination coefficient values (R2) for the linear fittings are shown, and data are presented as means ± standard deviations (n = 3). v0: initial velocity. AU: arbitrary units. appKM: apparent KM. appvmax: apparent maximal velocity. [S]0: initial substrate concentration.</p