A novel STK1-targeted small-molecule as an “antibiotic resistance breaker” against multidrug-resistant Staphylococcus aureus

Abstract

Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important “antibiotic-resistance-breaker,” which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.

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

“…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]. In L. monocytogenes, the broad-spectrum kinase inhibitor staurosporine, as well as some more selective repurposed human kinase inhibitors, was seen to inhibit PrkA activity and sensitize cells to b-lactam drugs [219,237].…”

“…Similarly, several studies have identified Stk1-inhibiting molecules from small-molecule libraries that sensitize MRSA S. aureus strains to b-lactams [223,[238][239][240]. Notably, Kant et al optimized a quinazoline-based Stk1-inhibiting molecule (Inh2-B1) that potentiates the bactericidal activity of the cephalosporins ceftriaxone and cefotaxime on drug-resistant S. aureus, both in vitro and in an in vivo septicemia mouse model with negligible cytotoxic side effects [240]. Mechanistically, Inh2-B1 was shown to reduce the expression of cell wall hydrolase-encoding genes important for cell wall synthesis and biofilm formation.…”

“…In L. monocytogenes, the broad-spectrum kinase inhibitor staurosporine, as well as some more selective repurposed human kinase inhibitors, was seen to inhibit PrkA activity and sensitize cells to b-lactam drugs [219,237]. Similarly, several studies have identified Stk1-inhibiting molecules from small-molecule libraries that sensitize MRSA S. aureus strains to b-lactams [223,[238][239][240]. Notably, Kant et al optimized a quinazoline-based Stk1-inhibiting molecule (Inh2-B1) that potentiates the bactericidal activity of the cephalosporins ceftriaxone and cefotaxime on drug-resistant S. aureus, both in vitro and in an in vivo septicemia mouse model with negligible cytotoxic side effects [240].…”

“…Mechanistically, Inh2-B1 was shown to reduce the expression of cell wall hydrolase-encoding genes important for cell wall synthesis and biofilm formation. This effect is likely to occur due to reduced phosphorylation and promoter binding of the WalR response regulator upon Stk1 inhibition [240]. Thus, in summary, several observations suggest that combined b-lactam and Hanks kinase inhibition therapy could represent a novel strategy to combat clinical b-lactam resistance.…”

The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus

“…Pentaglycine-lipid II interacts with the extracellular PASTA (penicillin binding protein and serine/threonine kinase associated) domains of Stk and triggers its kinase activity in vitro 9 . In fact, deletion of stp and stk in S. aureus causes cell division defects resulting in the formation of multiple and incomplete septa, differences in cell size and cell wall thickness 10 , 22 . In addition, stk and stk/stp deletion strains are more susceptible to cell wall-acting antibiotics like tunicamycin 12 , fosfomycin 12 , 20 and β-lactam antibiotics 10 , 16 .…”