For many years, we have tried to use antibiotics to eliminate the persistence of pathogenic bacteria. However, these infectious agents can recover from antibiotic challenges through various mechanisms, including drug resistance and antibiotic tolerance, and continue to pose a global threat to human health. To design more efficient treatments against bacterial infections, detailed knowledge about the bacterial response to the commonly used antibiotics is required. Proteomics is a well-suited and powerful tool to study molecular response to antimicrobial compounds. Bacterial response profiling from system-level investigations could increase our understanding of bacterial adaptation, the mechanisms behind antibiotic resistance and tolerance development. In this review, we aim to provide an overview of bacterial response to the most common antibiotics with a focus on the identification of dynamic proteome responses, and through published studies, to elucidate the formation mechanism of resistant and tolerant bacterial phenotypes.

Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signaling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well characterized Mps1 inhibitor, reversine. We show that estimates of the IC₅₀ (employing a novel specific and efficient assay that utilizes a fluorescently labeled substrate) and the binding affinity (K_D) indicate that, in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715 and compared the binding modes of Cpd-5, NMS-P715, and reversine. The difference in steric hindrance between Tyr/Trp⁶⁰⁴ and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro. Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.

Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signalling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well-characterised Mps1 inhibitor, reversine. We show that estimates of the IC ₅₀ (employing a novel specific and efficient assay that utilizes a fluorescently labelled substrate) and of the binding affinity ( K _D ) indicate that in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715, and compare the binding modes of Cpd-5, NMS-P715 and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro . Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises. Summary statement: The inhibition of specific Mps1 kinase inhibitors towards the wild-type protein and inhibitor-resistant mutants is explained by a novel specific activity assay, biophysical characterisation, and X-ray structures. Abbreviations: ATP adenosine triphosphate Bub1/Bub3 budding uninhibited by benzimidazoles 1 / budding uninhibited by benzimidazoles 3 Cpd-5 Compound-5 (N-(2,6-diethylphenyl)-8-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide) FP fluorescence polarization GST Glutathione S-transferase IC50 half maximal inhibitory concentration K _D dissociation constant ΔG _calc calculated Gibbs energy difference for ligand binding KNL1 kinetochore null protein 1 KPi (inorganic) potassium phosphate Mps1 monopolar spindle 1 MST microscale thermophoresis NMS-P715 N-(2,6-diethylphenyl)-1-methyl-8-({4-[(1-methylpiperidin-4-yl)carbamoyl]-2-(trifluoromethoxy)phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide TMR tetramethylrhodamine WT wild-type