Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

In order to confirm the stability of the peptides, them were diluted to 150 mM solution in double-distilled water, then incubated at 37 ºC for 24 hr. ESI-MS spectra for the various peptides are presented in Figure   S2 before the incubation ( Figure S2A) and after the incubation ( Figure S2B). All peptides besides pep1 were found to be stable at the given conditions.  In order to better visualize the effect of pep5, Figure S11 presents the transition from mostly live cells (green) to dead cells (red) in the increasing concentration of pep5. was suspended in 200 µl LB and cultured on a LB petri dishes. 2 mm radius discs were dipped into specified concentration of the peptide solution (peptide in 0.85% NaCl; sterile) and then placed in the Petri dishes.
Petri dishes were left at 37 C overnight. Figure S12A visualizes the differences in radius formed when different peptide-solution discs were placed in the Petri dish. Table S1 presents the resulting radiuses of bacteria killing. For control experiment, discs were dipped into various concentrations of kanamycin, the bacteria killing radius formed over the disks were measured and presented in Figure S5B.

Kanamycin [M]
A B

Cell toxicity (MTT assay)
Cell toxicity (MTT assay) experiments were conducted to verify the effect of pep5 on rat L6 myotubes.
Myotubes were cultured as described in Methods. Pep5 was introduced in various concentrations (three triplicates per concentration) ranging between 0-300 µM and incubated for 24 hr at 37 C. In addition, compound 8 as a positive control in concertation 100 µM was added. After then medium was taken out and 0.1% of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in PBS buffer was introduced. The myotubes were incubated for 2 hr at 37 C. The PBS buffer was collected from plates and 200 µl of DMSO were added and incubated with shaking for 30 min at room temperature. Lysates were transferred to 96 well plates and the absorption values were collected using Synergy plate reader at wavelength of 570 nm.
Calculations were based on the average of three repetitions. The absorption of the control sample without pep5 was determined as a 100%, all pep5 concentrations were compared against the control sample.

CW-EPR (Continuous-Wave EPR) measurements
CW-EPR spectra were recorded using an E500 Elexsys Bruker spectrometer operating at 9.0-9.5 GHz. The spectra were recorded at room temperature using a microwave power of 20.0 mW, a modulation amplitude of 1.0 G, a time constant of 60 ms, and a receiver gain of 60.0 dB. The samples were measured in 0.8 mm capillary quartz tubes (VitroCom). CusB was labeled at A236C and A248C (identical labeling as the DEER measurements) and measured both in the apo-state (Cu(I)-free) and in the holo-state (Cu(I)-bound) as well as the holo-state in the presence of pep5, pep8, and pep2. CusB presents significant changes in the hyperfine interaction (aN) upon Cu(I) binding which may be related to the conformational changes the protein undergoes in the environment surrounding the spin label. With the addition of pep5 the hyperfine interaction is similar to the apo-state of CusB indicating on the inhibition of those conformational changes. Pep8 also presented some changes in the hyperfine interaction, yet not as significant as pep5 did. For pep2 no major hyperfine interaction changes were observed compared with the holo-state of CusB.  Fig N: CW-EPR measurements. CW-EPR spectra of CusB (labeled at A236C and A248C) for the apo-state, holo-state (grey), apo-state and pep5, holo-state and pep5, holo-state and pep8, and holostate and pep2.