Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

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 < 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 >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.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

Wound natural healing in treatment of tumor-like hypertrophic scar.

BACKGROUND:: Surgical sutures, wound tension, additional skin incisions and other factors may result in recurrence of tumor-like scar. OBJECTIVE:: To investigate the role of wound natural healing therapy in tumor-like hypertrophic scar. METHODS:: In this study, tumor-like hypertrophic scars of 47 cases were excised completely and the residual wounds were treated with natural healing. The short-term and long-term effects of treatment were evaluated. RESULTS:: All cases were successfully cured by natural healing therapy. The healing time of the maximum wound (80mm × 20mm) and the minimal wound (5mm× 5mm) was 25 days and 7 days respectively. The size of new skin scars ranged from 3mm to 11 mm. Clinical followed-up was performed in 34 cases for 36 months. Among them, no recurrence happened in 31 cases and new scar size ranged from 2mm to 8mm, while local recurrence happened in 3 cases whose scar size were less than 5 mm. STUDY LIMITATIONS:: The cure rate of the therapy was 91.2%. CONCLUSION:: The wound natural healing therapy is effective in treating tumor-like hypertrophic scar, which can prevent recurrence and has good cosmetic results.

Wound natural healing in treatment of tumor-like hypertrophic scar

Abstract: Background: Surgical sutures, wound tension, additional skin incisions and other factors may result in recurrence of tumor-like scar. Objective: To investigate the role of wound natural healing therapy in tumor-like hypertrophic scar. Methods: In this study, tumor-like hypertrophic scars of 47 cases were excised completely and the residual wounds were treated with natural healing. The short-term and long-term effects of treatment were evaluated. Results: All cases were successfully cured by natural healing therapy. The healing time of the maximum wound (80mm × 20mm) and the minimal wound (5mm× 5mm) was 25 days and 7 days respectively. The size of new skin scars ranged from 3mm to 11 mm. Clinical followed-up was performed in 34 cases for 36 months. Among them, no recurrence happened in 31 cases and new scar size ranged from 2mm to 8mm, while local recurrence happened in 3 cases whose scar size were less than 5 mm. Study Limitations: The cure rate of the therapy was 91.2%. Conclusion: The wound natural healing therapy is effective in treating tumor-like hypertrophic scar, which can prevent recurrence and has good cosmetic results.