Optimized protocols for assessing libraries of poorly soluble sortase A inhibitors for antibacterial activity against medically-relevant bacteria, toxicity and enzyme inhibition

Formulation and Biological Evaluation of Mesoporous Silica Nanoparticles Loaded with Combinations of Sortase A Inhibitors and Antimicrobial Peptides

“…The results ( Figure 5 , Table 3 ) demonstrated that the MICs of SrtAI loaded MCM-41-PO 3 − MSNs were in most cases 2-fold lower than the SrtAI-loaded MCM-41 MSNs; the AMPs demonstrated potent antimicrobial activity against each tested bacterial strain, including MRSA, in the general order MASTO > PEX > INDO; and the unloaded MSNs did not demonstrate antimicrobial activity in the tested concentration range (≤500 mg/mL). In addition, the CUR and QC-loaded MSN formulations were demonstrated to be more potent than their previously reported 5% ( v / v ) DMSO Muller–Hinton Broth formulations [ 20 ], ranging from 4- to 16-fold improvements in their MIC values, with the MCM-41-PO 3 − MSN formulations in general being more potent. In comparison, a 2-fold maximum improvement was observed for TC and BR MCM-41-PO 3 − MSNs, with no improvement seen for TC and BR MCM-41 formulations.…”

“…A BMD assay, with a resazurin colorimetric readout [ 20 ], was performed to determine the MIC of each individual SrtAI-loaded ( Figure 5 ) and non-loaded MSN and AMP (PEX, INDO, and MASTO) ( Figure S4 ) against S. aureus (MSSA) ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853 ( Table 3 ). The results ( Figure 5 , Table 3 ) demonstrated that the MICs of SrtAI loaded MCM-41-PO 3 − MSNs were in most cases 2-fold lower than the SrtAI-loaded MCM-41 MSNs; the AMPs demonstrated potent antimicrobial activity against each tested bacterial strain, including MRSA, in the general order MASTO > PEX > INDO; and the unloaded MSNs did not demonstrate antimicrobial activity in the tested concentration range (≤500 mg/mL).…”

“…The MICs of each SrtAI-loaded MCM-41 and MCM-41-PO 3 − formulation against four bacterial strains ( S. aureus (MSSA) ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853) were determined using the CLSI BMD method [ 24 ] with a resazurin colorimetric readout [ 20 ]. For this method, quantities of SrtAI-loaded MCM-41 or MCM-41-PO 3 − containing 1 mg of individual SrtAIs were suspended in MHB (to 1 mL) to yield a 1 mg/mL final SrtAI concentration.…”

“…The MICs of PEX, INDO, and MASTO against S. aureus ATCC25923, MRSA ATCC43300, E. coli ATCC25922, and P. aeruginosa ATCC27853 were determined according to the CLSI BMD method with resazurin readout [ 20 ], as described above. In this method, 100, 200, and 300 µg of PEX, INDO, MASTO, respectively, were dissolved in 0.01% ( v / v ) DMSO-MHB (to 1 mL) and used to load column one of individual 96-well microplates.…”

“…Herein, in an attempt to overcome the solubility issues associated with each of the four SrtAI library compounds (TC, CUR, QC, and BR) through nanoencapsulation, improve their antimicrobial activity, and reduce their toxicity compared to formulations with organic co-solvents [ 20 ], the loading of anionic MCM-41 and phosphonate-modified MCM-41 (MCM-41-PO 3 − ) with each SrtAI was investigated, along with the effects of SrtAI loading on particle size, charge, morphology, and SrtAI aqueous solubility. It was hypothesized that these hydrophobic SrtAIs would be confined within the MCM-41 and MCM-41-PO 3 − pores in an amorphous form, helping to improve their aqueous solubility [ 21 ].…”