Structural Design and Synthesis of Novel Cyclic Peptide Inhibitors Targeting Mycobacterium tuberculosis Transcription

Abstract

Tuberculosis (TB) remains one of the deadliest infectious diseases in the world. Although several established antitubercular drugs have been found, various factors obstruct efforts to combat this disease due to the existence of drug-resistance (DR) TB strains, the need for lengthy treatment, and the occurrence of side effects from drug-drug interactions. Rifampicin (RIF) is the first line of antitubercular drugs and targets RNA polymerase (RNAP) of Mycobacterium tuberculosis (MTB). Here, RIF blocks the synthesis of long RNA during transcription initiation. The efficacy of RIF is low in DR-TB strains, and the use of RIF leads to various side effects. In this study, novel cyclic peptides were computationally designed as inhibitors of MTB transcription initiation. The designed cyclic peptides were subjected to a virtual screening to generate compounds that can bind to the RIF binding site in MTB RNAP subunit β (RpoB) for obtaining a new potential TB drug with a safe clinical profile. The molecular simulations showed that the cyclic peptides were capable of binding with RpoB mutants, suggesting that they can be possibility utilized for treating DR-TB. Structural modifications were carried out by acetylation and amidation of the N- and C-terminus, respectively, to improve their plasma stability and bioavailability. The modified linear and cyclic peptides were successfully synthesized with a solid-phase peptide synthesis method using Fmoc chemistry, and they were characterized by analytical HPLC, LC-ESI-MS⁺, and 1H NMR.

Keywords: cyclic peptides; molecular simulation; solid-phase peptide synthesis; tuberculosis.

Figure 1

RRDR region of MTB RpoB. (A) Visualization of MTB RpoB with RIF binding site shown in a circle. (B) The RDRR shown in brown ribbon relative to the RIF molecule on the RIF binding site.

Figure 2

Detailed amino acid labels at RIF binding site on RpoB. The purple and green surfaces indicate hydrophilic and hydrophobic areas, respectively.

Figure 3

3D Interactions of (A) (Cyclo-1,6)CYYQWC, (B) (Cyclo-1,6)CFSRMC, (C) (Cyclo-1,6)CLYHFC, (D) (Cyclo-1,6)CYTYWC, (E) (Cyclo-1,6)CLYKVC, and (F) RIF in the RpoB WT binding pocket as shown in the grey skeletal model. The blue area indicates the hydrophilic region, while the red area designates the hydrophobic region on the binding site.

Figure 4

Protein–ligand interactions of (A) (Cyclo-1,6)CYYQWC, (B) (Cyclo-1,6)CFSRMC, (C) (Cyclo-1,6)CLYHFC, (D) (Cyclo-1,6)CYTYWC, and (E) (Cyclo-1,6)CLYKVC in the binding sites of RpoB WT (left panel) and S450L mutant (right panel) from a flexible molecular docking simulation. Hydrophobic and hydrophilic amino acid residues are represented in the green and purple circles, respectively. Interactions, such as hydrogen bond and arene–hydrogen, are represented by arrows, and ion contact is represented by purple dashed lines.

Figure 5

The cyclization effect, as follows: (A) 3D Protein–ligand interactions of linear-CYTYWC (cyan) and (Cyclo-1,6)CYTYWC (magenta), and (B) 2D interaction of linear-CYTYWC and (Cyclo-1,6)-CYTYWC with RIF binding site on RpoB. Hydrophobic and hydrophilic amino acids residues are represented in green and purple circles, respectively. Interactions, such as hydrogen bond and arene–hydrogen, are represented by arrows, and ion contact is represented by purple dashed lines.

Figure 6

Synthetic scheme of (Cyclo-1,6)Ac-CYYQWC-NH₂ with Rink amide MBHA resin: (a) Fmoc deprotection: piperidine 20% in DMF; (b) addition of Fmoc-L-Cys(Trt)-OH and Fmoc-L-Trp(Boc)-OH. Fmoc-L-Gln(Trt)-OH, Fmoc-L-Tyr(tBu)-OH (2×), Fmoc-L-Cys(Trt)-OH with HCTU/NMM in DMF as the coupling reagent; (c) final deprotection with 20% piperidine in DMF, followed by acetylation to the N-terminus with acetic anhydride and 2.6-lutidine in DMF, RT; (d) resin cleavage and global deprotection: TFA/TIPS/Phenol/H₂O (90:5:2.5:2.5), RT, 2 h reaction, followed by crude precipitation and RP-HPLC purification resulted in the linear-Ac-CYYQWC-NH₂; and (e) cyclization with air dilution in a high dilution of ammonium bicarbonate buffer pH = 8.5, followed by characterization.