Optimization of a Benzothiazole Indolene Scaffold Targeting Bacterial Cell Wall Assembly

Drug Design, Development and Therapy Dovepress open access to scientific and medical research Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 125.142.225.230 on 14-Jul-2020 For personal use only. Open Access Full Text Article Optimization of a Benzothiazole Indolene Scaffold Targeting Bacterial Cell Wall Assembly This article was published in the following Dove Press journal: Drug Design, Development and Therapy Jay Chauhan 1 Wenbo Yu 2 Steven Cardinale 3 Timothy J Opperman 3 Alexander D MacKerell Jr 2,4 Steven Fletcher 1 Erik PH de Leeuw 1 1 Institute of Human Virology & Department of Biochemistry and Molecular Biology of the University of Maryland Baltimore School of Medicine, Baltimore, MD 21201, USA; 2ComputerAided Drug Design Center, University of Maryland, School of Pharmacy, Baltimore, MD 21201, USA; 3Microbiotix, LLC., Worcester, MA 01605, USA; 4 Department of Pharmaceutical Sciences, University of Maryland, School of Pharmacy, Baltimore, MD 21201, USA Correspondence: Erik PH de Leeuw Tel +1 410 706 3430 Email Background: The bacterial cell envelope is comprised of the cell membrane and the cell wall. The bacterial cell wall provides rigidity to the cell and protects the organism from potential harmful substances also. Cell wall biosynthesis is an important physiological process for bacterial survival and thus has been a primary target for the development of antibacterials. Antimicrobial peptides that target bacterial cell wall assembly are abundant and many bind to the essential cell wall precursor molecule Lipid II. Methods: We describe the structure-to-activity (SAR) relationship of an antimicrobial peptide-derived small molecule 7771–0701 that acts as a novel agent against cell wall biosynthesis. Derivatives of compound 7771–0701 (2-[(1E)-3-[(2E)-5,6-dimethyl-3-(prop2-en-1-yl)-1,3-benzothiazol-2-ylidene]prop-1-en-1-yl]-1,3,3-trimethylindol-1-ium) were generated by medicinal chemistry guided by Computer-Aided Drug Design and NMR. Derivatives were tested for antibacterial activity and Lipid II binding. Results: Our results show that the N-alkyl moiety is subject to change without affecting functionality and further show the functional importance of the sulfur in the scaffold. The greatest potency against Gram-positive bacteria and Lipid II affinity was achieved by incorporation of a bromide at the R3 position of the benzothiazole ring. Conclusion: We identify optimized small molecule benzothiazole indolene scaffolds that bind to Lipid II for further development as antibacterial therapeutics. Keywords: Lipid II, antibiotics, drug development, cell wall Cell wall biosynthesis is a complex process that occurs in three stages: the cytoplasmic stage, the membrane-associated stage and at the cell wall envelope.1,2 The main purpose of this process is the translocation of the peptidoglycan subunits N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) from the cytoplasm across the cellular membrane. These two amino sugars are coupled together by β-1,4-glycosidic bonding. Cross-linking of glycan chains occurs via amide to backbone bond formation of a pentapeptide moiety, which is attached to the MurNac sugar.2–5 Lipid II (GlcNAcMurNAc(pentapeptide) phosphoryl undecaprenol) is an essential intermediate in cell wall biosynthesis. Since Lipid II is partly accessible to the extracellular compartment of the cytoplasmic membrane, it is a target for antibacterial compounds.2,5 These compounds include glycopeptides that have been in clinical use, such as vancomycin, as well as other classes of antibacterial peptides like lantibiotics, ramoplanins and defensins.2,6–9 Based on the interaction between Lipid II and the antimicrobial peptide Human Neutrophil Peptide −1 (HNP-1),9 we identified low molecular weight synthetic compounds that target Lipid II with high specificity and affinity.10 In this study, we report on the structural and functional relationships of derivatives of lead compound 7771–0701. 567 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2020:14 567–574 DovePress © 2020 Chauhan et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://doi.org/10.2147/DDDT.S226313 Powered by TCPDF (www.tcpdf.org) ORIGINAL RESEARCH Dovepress Chauhan et al Drug Design, Development and Therapy downloaded from https://www.dovepress.com/ by 125.142.225.230 on 14-Jul-2020 For personal use only. Materials and Methods CADD Modeling and MD Simulations Molecular Dynamics (MD) simulations were performed with the program CHARMM11 using the CHARMM36 lipid12 and protein force field13,14 for Lipid II, the CHARMM TIP3P model15 for water, along with the CHARMM General force field16–18 for the ligand. Based on the previously published model from MD simulation for the BAS00127538-Lipid II complex system in aqueous solution,10 the lead compound 7771–0701 was docked onto Lipid II by aligning its indolene group with that of BAS00127538. The system was then subjected to a short energy minimization following which a 100 ps MD simulation with a time step of 0.5 fs was carried out to further equilibrate the system. The system was then subjected to a 20 ns MD simulation run with a time step of 1 fs. Simulations were carried out in the NPT ensemble at 300 K and 1 atm with SHAKE of covalent bonds involving hydrogens, and there were no restraints in the simulations. Free energies of binding, ΔG, were estimated using the linear interaction energy (LIE) method,19 where elec ΔG ¼ α Eelec bound  Eunbound vdw þ β Evdw bound  Eunbound
þγ (Eq:1) in which α = 0.5, β = 0.16, γ cancels out as we only considered the relative free energies ΔΔG, and the unbound interaction energies were computed from 5 ns MD simulations of the compound alone in water. This involved manually placing one of the inhibitor benzene rings and MurNac ring in Lipid II adjacent to each other. Harmonic restraints, k(r-r0)2, were placed between the geometric centers of the above groups, where k=50 kcal/ (mol Å2), r0=3 Å and r is the distance between those geometric centers. The system was then subjected to a 2000 step SD energy minimization followed by a 1 ns gas phase Langevin simulation in the presence of the restraints followed by an additional 1 ns gas phase Langevin simulation in the absence of the restraints. The resulting complex was then solvated in a 48*48*48 Å3 pre-equil (...truncated)