Dual‐Acting Small‐Molecule Inhibitors Targeting Mycobacterial DNA Replication

Abstract

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium and a causative agent of tuberculosis (TB), a disease that kills more than 1.5 million people worldwide annually. One of the main reasons for this high mortality rate is the evolution of new Mtb strains that are resistant to available antibiotics. Therefore, new therapeutics for TB are in constant demand. Here, we report the development of small‐molecule inhibitors that target two DNA replication enzymes of Mtb, namely DnaG primase and DNA gyrase (Gyr), which share a conserved TOPRIM fold near the inhibitors’ binding site. The molecules were developed on the basis of previously reported inhibitors for T7 DNA primase that bind near the TOPRIM fold. To improve the physicochemical properties of the molecules as well as their inhibitory effect on primase and gyrase, 49 novel compounds have been synthesized as potential drug candidates in three stages of optimization. The last stage of chemical optimization yielded two novel inhibitors for both Mtb DnaG and Gyr that also showed inhibitory activity toward the fast‐growing non‐pathogenic model Mycobacterium smegmatis (Msmg).

Cell-penetrating peptide conjugates of indole-3-acetic acid-based DNA primase/Gyrase inhibitors as potent antitubercular agents against planktonic and biofilm culture of Mycobacterium smegmatis

“…Importantly, these enzymes in prokaryotes share common TOPRIM fold that constitute the side wall of the binding-site cleft of the small molecule inhibitors we found. Using NMRfragment based screening and computational optimization we found novel indole-3-acetic acid derivatives that inhibit T7 primase but also DnaG and Gyrase of Mtb (Ilic et al, 2016;M. Singh et al, 2020).…”

“…The copyright holder for this this version posted December 1, 2020. ; https://doi.org/10.1101/2020.12.01.406405 doi: bioRxiv preprint RNA primer synthesis was reported previously (M. Singh et al, 2020) (Table 2). The copyright holder for this this version posted December 1, 2020. ; https://doi.org/10.1101/2020.12.01.406405 doi: bioRxiv preprint 8 5'-CCGACCCGTCCGTAATACAGAGGTAATTGTCACGGT-3', α-32P-ATP, CTP, GTP, UTP in a standard reaction mixture, and different concentrations in double dilutions of each small molecule.…”

“…The copyright holder for this this version posted December 1, 2020. ; https://doi.org/10.1101/2020.12.01.406405 doi: bioRxiv preprint 6 Singh et al, 2020). Next, we wanted to explore if the inhibitory activity would be improved upon conjugating these molecules to a peptide carrier.…”

“…The design, synthesis, and optimization of DNA/Gyrase inhibitor (IN) were reported in our previous work(M. Singh et al, 2020).Within the series reported in our previous work we have selected most active analogue (IN) for conjugations. The characterization of the compound IN is provided in the supporting information (ESI).…”

Development of Single‐Stranded DNA Bisintercalating Inhibitors of Primase DnaG as Antibiotics

“…These initial studies found low‐micromolar inhibitors of DnaG that did not have antibacterial activity, [6] which was likely the reason for the lack of their further development. Another recent study identified very weak (∼100 μM) DnaG inhibitors [7] . In the high‐micromolar/low‐millimolar concentration range, where such small molecules display the activity of interest, they are expected to bind multiple low‐affinity targets and require a 100‐fold or greater improvement in potency by synthetic methods, which is neither likely nor practical.…”