In Silico Screen and Structural Analysis Identifies Bacterial Kinase Inhibitors which Act with β-Lactams To Inhibit Mycobacterial Growth

Abstract

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like ser/thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modelling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel x-ray structure confirmed that the inhibitors bind as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity towards Mycobacteria and Nocardia. We demonstrated the inhibitors are uniquely potentiated by β-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB’s role in sensing cell wall stress. This is the first demonstration in the phylum Actinobacteria that some β-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Their in silico structural docking revealed a family of imidazopyridine aminofurazan lead compounds with high binding affinity toward PknB. Intriguingly, and consistent with the known role of PknB in cell wall homeostasis, these inhibitors were able to significantly potentiate the effect of b-lactams in several types of mycobacteria and in the related Nocardia asteroides [236]. This synergistic concept has also been reported for kinase inhibitors of the nonessential L. monocytogenes PrkA and S. aureus Stk1 Hanks kinases [219,223,[237][238][239][240].…”

“…Another approach to identify kinase inhibitors is by in silico modeling. Wlodarchak et al [236] exploited the general structural similarity of bacterial and human Ser/ Thr kinases to identify PknB inhibitors by in silico screening of a human kinase inhibitor collection. Their in silico structural docking revealed a family of imidazopyridine aminofurazan lead compounds with high binding affinity toward PknB.…”

NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G

“…Analysis of the induced-fit docking poses of NU-6027 in PknB showed that the functional residues, such as Glu93 and Val95, are not involved in hydrogen bond formation with NU-6027. Previously, it has been shown that an inhibitor, G93, forms a hydrogen bond with residues such as Glu59, Lys40, and Asp156 (32). However, these residues are not involved in hydrogen bond formation in the predicted PknB:NU-6027 induced-fit docked poses (Fig.…”

“…The kinase domain in 2PZI is reported with the DFG loop of the kinase domain in the "in" conformation (31); the information on the conformational state of the kinase domain in 5U94 (32) is not explicitly available. Superimposition of the kinase domain from different crystal structures of ligand-bound PknB complexes (1MRU, 1O6Y, and 2FUM) onto the kinase domain of PknB in 5U94 suggested that the DFG loop in the latter is in the "in" conformation (29,30,32,75). This pairwise superimposition was performed using the Protein Structure Alignment module in Maestro, version 11.01.011 (Schrodinger LLC).…”

Protein kinases PknA and PknB independently and coordinately regulate essential Mycobacterium tuberculosis physiologies and antimicrobial susceptibility

“…Deletion or inhibition of PknB orthologues in Gram-positive bacteria can lead to increased susceptibility to β-lactam antibiotics [34][35][36], and meropenem has been shown to potentiate the activity of a PknB inhibitor 2-fold in auxotrophic M. tuberculosis [37]. Though carbapenems have been used to treat MDR-TB, cephalosporins and penicillins are generally not active against M. tuberculosis at clinically relevant concentrations.…”