The ClpB/DnaK bi-chaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials, because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an anti-tumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd~60 μM and inhibits the casein-activated, but not the basal ATPase activity of ClpB with an IC50~5 μM. The remaining p97 ligands, which displayed a higher affinity towards p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd~100 μM, did not affect the DnaK ATPase, but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB/DnaK bi-chaperone system in vitro with an IC50~5 μM and suppressed the growth of cultured E. coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock, but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ towards ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.

A molecular chaperone ClpB disaggregates and reactivates aggregated proteins in cooperation with DnaK, DnaJ, and GrpE. Within a cellular environment, ClpB must distinguish between properly folded and aggregated proteins by recognizing specific physical and/or chemical surface properties of the aggregates. However, the molecular mechanism of substrate binding to ClpB is poorly understood. We hypothesized that ClpB recognizes those polypeptide segments that promote protein aggregation because they are likely present at the surface of growing aggregates. We used an algorithm TANGO (Fernandez-Escamilla et al., Nat. Biotech. 2004, 22, 1302) to predict the aggregation-prone segments within the model ClpB-binding peptides and investigated interactions of the FITC-labeled peptides with ClpB using fluorescence anisotropy. We found that ClpB binds the substrate-mimicking peptides with positive cooperativity, which is consistent with an allosteric linkage between substrate binding and ClpB oligomerization. The apparent affinity towards ClpB for peptides displaying different predicted aggregation propensities correlates with the peptide length. However, discrete aggregation-prone segments within the peptides are neither sufficient nor necessary for efficient interaction with ClpB. Our results suggest that the substrate recognition mechanism of ClpB may rely on global surface properties of aggregated proteins rather than on local sequence motifs.

Aims: To evaluate antibacterial activity of aqueous, methanol, dichloromethane, and hexane extracts of Artocarpus nobilis Thw. leaves and stem bark against Escherichia coli and Staphylococcus aureus using agar well diffusion method. Methodology: Matured fully expanded leaves and stem bark parts of Artocarpus nobilis were collected, air-dried, and grounded. The extraction was obtained using a decoction extraction method. Antibacterial activity was performed against Staphylococcus aureus (ATCC® 25923TM) and Escherichia coli (ATCC® 25922TM) using agar well diffusion method and gentamicin was used as a positive control. The whole experiment was done in triplicates and the diameter of the inhibition zone (in mm) was measured and recorded. Results: Results showed that aqueous bark extract (EC50 4.286 mg/mL) showed the highest efficacy and potency against E. coli while methanol bark extract (EC50 4.427 mg/mL) showed the highest efficacy and potency against S. aureus. R² and P values for aqueous, methanol, dichloromethane, and hexane extracts indicated that there was a strong, statistically significant correlation (P ≤ 0.05) between concentration and zone of inhibition for all extracts of A. nobilis against E. coli and S. aureus. Conclusion: This study showed that aqueous and methanol bark extracts of Artocarpus nobilis have marked in vitro dose-dependent antibacterial activity against E. coli and S. aureus respectively. Further studies are necessary to ascertain the mechanism and the active constituents responsible for the antibacterial activity of the of plant parts of Artocarpus nobilis.

Binding configuration of a de novo stapled peptide on SARS-CoV-2 spike protein, as predicted by molecular simulation. Stapled residues enhance peptide stability while interacting residues engage key amino acids on the protein receptor-binding domain.

Aims: To investigate in vitro anti-inflammatory activity of aqueous, methanol, dichloromethane, and hexane extracts of Artocarpus nobilis Thw. leaves and stem bark using heat-induced protein denaturation test (egg albumin denaturation). Methodology: About 500 g of each matured, fully expanded leaves and stem bark of Artocarpus nobilis were collected, washed and air-dried. Leaves and stem bark parts were grounded to obtain a fine powder material. The extractions were obtained using the decoction extraction method. Anti-inflammatory activity was evaluated using the heat-induced egg albumin denaturation method. Diclofenac sodium was used as the positive control. Results: Results showed that Diclofenac sodium exhibited an IC50 value of 243.4 µg/mL and methanolic stem bark extract had an IC50 Value of 249.8 µg/mL) for heat-induced egg albumin protein denaturation test. R² and P values for aqueous, methanol, dichloromethane, and hexane extracts indicated that there was a strong, statistically significant correlation (P<0.01) between concentration and percentage inhibition for all extracts of A. nobilis Thw. However, methanol stem bark extract demonstrated the highest efficacy and potency with similar activity observed for the positive control Diclofenac sodium. Conclusion: Methanol stem bark extract of Artocarpus nobilis Thw. have marked in vitro anti-inflammatory activity. Further studies are necessary to determine the mechanism and the active constituents responsible for the anti-inflammatory activity of the plant parts of Artocarpus nobilis Thw.