New Chemotypes for the Inhibition of (p)ppGpp Synthesis in the Quest for New Antimicrobial Compounds

Abstract

Antimicrobial resistance (AMR) poses a serious threat to our society from both the medical and economic point of view, while the antibiotic discovery pipeline has been dwindling over the last decades. Targeting non-essential bacterial pathways, such as those leading to antibiotic persistence, a bacterial bet-hedging strategy, will lead to new molecular entities displaying low selective pressure, thereby reducing the insurgence of AMR. Here, we describe a way to target (p)ppGpp (guanosine tetra- or penta-phosphate) signaling, a non-essential pathway involved in the formation of persisters, with a structure-based approach. A superfamily of enzymes called RSH (RelA/SpoT Homolog) regulates the intracellular levels of this alarmone. We virtually screened several fragment libraries against the (p)ppGpp synthetase domain of our RSH chosen model RelSeq, selected three main chemotypes, and measured their interaction with RelSeq by thermal shift assay and STD-NMR. Most of the tested fragments are selective for the synthetase domain, allowing us to select the aminobenzoic acid scaffold as a hit for lead development.

The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives

“…The Rel Mtb crystal structure unbound to the substrate [ 57 ] was used to analyze the ligand binding to an inactive state synthetase, while the Rel Seq structure complexed with a GDP (guanosine diphosphate) substrate [ 58 ] was used to investigate the binding to (p)ppGpp synthetase in a catalytically competent state. The reference ligand indole-5-carboxylic acid demonstrated a Rel Seq active site binding energy of −6.42 kcal/mol ( Table 4 ), consistent with the result of −6.41 kcal/mol calculated in the previous research (in which ligand mentioned as I2) [ 26 ].…”

“…Searching for new chemical compounds that are targeted to non-essential bacterial adaptation pathways, such as stringent response, is an attractive approach for developing antimicrobials preventing bacterial persistence, biofilm formation, quorum sensing, and virulence [ 56 ]. Previously, researchers have identified indole derivatives as a chemotype capable of binding the Rel Seq (p)ppGpp synthetase/hydrolase synthetic domain via virtual screening and thermal shift assays [ 26 ]. Based on these data, we selected a set of substituted indoles from our library of compounds to assess the possibility of their binding to Rel Mtb or Rel Seq proteins in synthetic domain (SYN) active sites by using molecular docking methods.…”

“…In turn, the inhibition of these enzymes suppressed the ability of bacterial cells to form a persister state responsible for antibiotic tolerance. Furthermore, it was shown that substituted indoles are able to bind Rel Seq (p)ppGpp synthetase/hydrolase and can constitute a promising scaffold for (p)ppGpp synthetase enzyme inhibitors’ development [ 26 ].…”

“…Herein, we describe a straightforward approach to 2-(1 H -indol-3-yl)quinazolin-4(3 H )-one and their analogues based on the condensation of anthranilamides with aldehydes and an investigation of their cytotoxicity and antibacterial activity, including tests against Mycobacterium tuberculosis H 37 Rv, as well as their influence on the formation of biofilms. We describe the results of molecular docking studies on the ability of substituted indoles to bind to long RSH proteins, using two proteins functioning as the alarmone synthetases: Rel Mtb from Mycobacterium tuberculosis [ 41 ] and Rel Seq from Streptococcus equisimilis as the models [ 26 ].…”

Women in Bioorganic Chemistry

“…Sattin and co-workers from Milano, Italy, described, through virtual screening accompanied by STD-NMR studies, a structure-based approach to find new chemotypes able to target (p)ppGpp (guanosine tetra-or penta-phosphate) signaling, in view of overcoming antimicrobial resistance [9]. The issue of antimicrobial resistance was also addressed by the group of Grosdemange-Billiard and co-workers from Strasbourg, France, who synthesized fluorinated analogues of the natural compound fosmidomycin and tested them as E. coli 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) inhibitors as well as antimicrobial agents against E. coli on Petri dishes [10].…”