Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound

Abstract

The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈ 45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

Figure 1. Intermolecular interactions between PLASMEPSIN- II and Protease inhibitor of marine actinobacteria A. S. albogriseolus PI; B. S. longisporus PI; C. S. fradiae PI; D. S. lividans; E. S. griseus.

Enzyme PLASMEPSIN-II (indicated in red color) and their corresponding inhibitors (indicated in green color) are represented in ribbon configuration.

Figure 2. Streptomyces sp LK3 strain showed high protease inhibitor activity against trypsin and chymotrypsin with IC₅₀ values of LK3 strain A. trypsin (96.77 µg/ml) and B. chymotrypsin (161.8 µg/ml) B.

Mode of protease inhibition by peptide. The double-reciprocal plot displayed non-competitive inhibition mode C. Reversibility of peptide action was confirmed by the continuous steep decline in Vmax with an increase in inhibitor concentration with Km value remaining confined.

Figure 3. The 16s RNA sequencing confirmed the strain LK3 is a novel species with G+C content of the isolated DNA at 57.29 mol%.

Figure 4. Purification of protease inhibitor.

A. Elution pattern of protease inhibitor on DEAE-Sepharose anion exchange column; B. Elution pattern of protease inhibitor on C18-RP HPLC column; C. LCMS analysis of purified compound

Figure 5. Characterization of protease inhibitor.

A. Mass spectrum of peptide compound obtained from the GCMS clearly revealed that it may be a small peptide with 5 or 6 aminoacids (Molecular Weight-568da) The amino acids may be present in the peptide are: AILKRVMGNC; B. FTIR spectrum of peptide compound. Absorbance at 1632 corresponded to the carbonyl functions whereas the absorbance from 3300–3600cm-1 may be due to the NH function from the aminoacid functional groups.

Figure 6. Characteristics of PbA infection in Swiss Albino Mice and effect of LK1 (800 mg/kg/BW), LK2 (1000 mg/kg/BW) and LK3 (600 mg/kg/BW) respectively.

(A) Resulting parasitemias were expressed as a percent parasitemia (mean ± SD), measured daily on Giemsa stained blood smears. Parasitemia was evaluated statistically by Two-way ANOVA with Bonferroni post-test showed that the parasitemias were significantly different (*p<0.05) between PbA infected groups and LK3 treated groups, whereas parasitemias were not significantly different in the LK1, LK-2 group w.r.t. PbA infected controls and the matched controls (P>0.05). (B) Effect of LK1 (800 mg/kg/BW), LK2 (1000 mg/kg/BW) and LK3 (600 mg/kg/BW) respectively on survival of mice infected with P. berghei ANKA. Each value in Y axis represents the percentage (%) of survival of the treated groups compared to PbA treated control mice. The treatment groups represent mice injected intraperitoneally from 2 dpi till the last day of survival. Results are presented as arithmetic mean (±SE) of five mice per group. (C) Percentage parasitemia suppression after administration of LK1 (800 mg/Kg), LK2 (1000 mg/Kg) and LK3 (600 mg/Kg) respectively. Data from five replicates are presented as arithmetic mean ± standard deviation and indicate p<0.05 with respect to the controls. Data shown are one representative experiment of five (n).

Figure 7. Histopathological changes in hepatic and splenic tissues in response to LK3 treatment during PbA infection.

Hematoxylin and eosin stains were used to prepare sections of the liver and spleen from mice treated with vehicle, PbA infected + 600 mg/Kg LK3 and PbA infected respectively. Sections of control (A) and PbA+LK3 infected (B) dpi liver showing, surrounding hepatocytes with nuclei and blood sinusoids lined with Kupffer cells. Liver from infected mice (C) shows, moriform vacuolization of hepatocytes and hemozoin pigmented Kupffer cells on 8 dpi. In control (D) and PbA+ LK3 infected spleen tissue section (E), distinct and defined separate cluster of white pulp marked by yellow arrows. In treatment tissue sections white pulp and red pulp are not distinct (F). Appearances of hollow spaces without cells observed in spleen tissue sections compared to that of control (red arrows). Black arrows represent hemozoin accumulation in liver and spleen tissue sections. Magnification indicated 40×

Figure 8. Cell lysate from respective control, treatment spleen and liver were subjected to western blot analysis and ELISA (A) liver and (B) spleen during PbA infection and treated with peptide (C) ELISA results (p<0.05, ANOVA followed by post hoc LSD test).

Figure 9. Possible mechanism of peptide from Streptomyces sp LK3 against malaria.