Molecular Modelling Modelling was performed using UCSF chimera [26]

Molecular Modelling Modelling was performed using UCSF chimera [26]. such as pneumonia, septicemia, and meningitis, but its susceptibility to existing classes of antimicrobials is also on a decline [2]. mediates disease through a wide range of well-characterized virulence factors such as pneumolysin to facilitate colonization, nutrient scavenging, and immunoevasion [3]. Commonly-used bacteriolytic antimicrobials such as -lactams have often been criticized for their role in the undesired elevation of toxin levels into host environments and affecting the treatment outcome of infections [4]. The identification of unprecedented targets is crucial to the discovery of novel antimicrobial agents for treatment against infections caused Cdc14A1 by RNAP holoenzyme SB-3CT (PDB: 4LJZ; 27) with the RNA polymerase core enzyme colored in gray, the CH region in yellow and 70 in blue. (B) The interaction between RNAP CH region (surface in mesh) and the N-terminal domain of 70 (blue helix) with key amino acid residues labeled. (C) C3 docked in the pharmacophore model. Green spheres, H-bond acceptors; pink spheres, H-bond donor; cyan spheres, hydrophobic groups. (D) The docking model C3 (left) and C3-005 (right) with the CH region in yellow helix and mesh surface. The interaction between RNAP and factors has been considered a target for novel antimicrobial discovery [9,10,11,12,13] as opposed to other inhibitors which target RNAP enzyme activities (such as rifampicin SB-3CT binding near the active site, lipiarmycin leading to allosteric inhibition of template DNA binding, myxopyronin and squaramides blocking the switch region of SB-3CT the RNAP clamp open-close) [5]. Previously, by rational design and pharmacophore model-based screening, we have identified three chemical compounds (Figure 2) that inhibit bacterial RNAP- interaction by binding to the CH region of RNAP [14]. One of the three compounds (C5), composed of a steroidal ABC tricyclic ring and an indolone moiety which commonly appear in natural products, was chosen for characterization. C5 was shown to inhibit RNAP- interaction in an ELISA-based assay as well as an transcription assay [14]. C5 demonstrated mild bacterial growth inhibition against both Gram-positive and Gram-negative screening. 2. Results and Discussion 2.1. Docking Study of C3 and Its Antimicrobial Activity We are particularly interested in C3 as it is a small molecule with drug-like properties predicted by Discovery Studio 2016 (Biovia, San Diego, California, United States). The substituted benzene rings can be easily modified and are suitable for studying the structure-activity relationship and validating our previously established pharmacophore models (Figure 1C). Nevertheless, modifications may be made to improve the inhibitory and antimicrobial activity of C3. As shown in the docking model (Figure 1D, left), C3 fits into the pharmacophore model using the right benzoic acid to form an ionic bond as the key anchor to R278 or R281 of RNAP CH, while the left substituted benzene ring may form interactions with I291 of RNAP CH by van der Waals forces, which is appropriate for an initial modification to probe the interaction with RNAP CH and identify a lead compound for further studies. The antimicrobial activities of C3 were first tested to determine the minimum inhibitory concentration (MIC) in accordance with the guidelines published by the CLSI using six bacterial species from the most recent WHO priority pathogens list for guiding R&D of new antibiotics consisting of three Gram-positive and three Gram-negative bacteria: spp. [15]. C3 shows very mild antimicrobial activity (MIC 256 g/mL) against ATCC 49619 (Table 1). Table 1 Antimicrobial activity of C3 and derivatives. ATCC 19433, SAURa: ATCC 25923, SAURb: ATCC 29213, SPNE: ATCC 49619, ABAU: ATCC 19606, PAER: ATCC 27853, ECLO: ATCC 13047, ECOL: ATCC 25922, VAN: vancomycin, RIF: rifampicin. 2.2. Molecular Mechanism of C3 by Inhibiting the Protein-Protein Interaction between RNAP CH- We then confirmed the mechanism of C3 at the molecular level by assessing SB-3CT the inhibition against the protein-protein interaction (PPI) at the major binding site between RNAP CH region and . Previously established split-luciferase assay was employed [16], in which the CH region of RNAP (amino acid 220-315) and full-length A were each tagged with one of the luciferase complementation fragments. In the absence of inhibitors, the interaction between CH-facilitates the reformation of the luciferase indicated by the luminescence released. Reduction of the luminescence signal due to inhibitor treatment reflects the percentage of inhibition of the PPI between CH- as compared to the control without inhibitor. As a result, the IC50 of C3 against the PPI between CH- at.