Efficacy and Toxicity Studies of Novel α-MSH Analogues with Antibiofilm Action and β-Lactam Resensitization Potential against MRSA

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), a biofilm-forming recalcitrant pathogen with a multidrug-resistant profile, poses a pandemic threat to human health and is the leading cause of severe infections in both healthcare and community settings. In this study, toward designing novel α-MSH-based peptides with enhanced activity and stability against MRSA, particularly its stationary phase and biofilm, we explored a design approach to augment the hydrophobicity of an 8-mer C-terminal α-MSH(6-13)-based peptide Ana-5 through the incorporation of a bulky unnatural amino acid. The designed Ana-peptides overcame the limitation of diminished activity in biological media and exhibited enhanced antistaphylococcal activity and cell selectivity. With membrane rupture as the primary mode of action, the peptides exhibited inhibitory potential against S. aureus biofilms. Importantly, the peptides did not exhibit any adverse effects in the in vivo toxicity studies and were also able to significantly alleviate bacterial infection in a systemic infection mice model study. Additionally, the peptides retained their activity in the presence of serum and displayed a low propensity toward resistance development in MRSA cells. Moreover, the observed synergistic potential of Ana-10 with conventional antibiotics could be vital in resurrecting discarded antibiotics. Thus, this study provides us with an exciting lead, Ana-10, for further development against biofilm-based chronic S. aureus infections.

Synergistic Potential of α-Melanocyte Stimulating Hormone Based Analogues with Conventional Antibiotic against Planktonic, Biofilm-Embedded, and Systemic Infection Model of MRSA

“…For Ana-10 (Table 3), the combination with oxacillin showed strong synergy against MRSA ATCC 33591, in line with our previous report. 26 Interestingly, similar to Ana-9, against MRSA ATCC 43300, Ana-10 also exhibited synergy with the same three β-lactam antibiotics viz. oxacillin, ampicillin, and cephalothin.…”

“…Previously, we reported the in vivo efficacy of the designed peptides, which showed 1−2 log reduction (∼3−20% survival) in the MRSA load in liver and kidney upon administration of Ana-9/Ana-10 at 250 μg/kg of body weight while the comparator antibiotic, oxacillin at 2 mg/kg, did not elicit any significant effect on the bacterial load. 26 As seen from Figure 3(A), upon increasing the concentration to 1 mg/kg, Ana-9 caused ∼4−5 log reduction in the bacterial load compared to the infection control in both kidney (p < 0.001) and liver (p < 0.05). Similarly, Ana-10 Encouragingly, the treatment of infected mice with an antimicrobial cocktail of Ana-9 at 250 μg/kg of body weight and oxacillin at 2 mg/kg of body weight exhibited in vivo synergistic anti-MRSA activity with a significant (p < 0.05) ∼4−5 log reduction of bacterial load in both kidney and liver, and the effect was comparable to the treatment at 1 mg/kg of the peptide alone.…”

“…The observed synergistic effect might be attributed to the simultaneous action of oxacillin, which weakens the peptidoglycan layer of the cell wall, and the membrane active Ana-peptide, which rapidly depolarizes and permeabilizes the membrane, resulting in a cascade of membrane and cell wall disintegration and further limiting the cell wall synthesis (repair) process leading to complete disintegration of the bacterial membrane, and ultimately the death of bacteria. 26,27,31,32 In addition, studies have shown that the permeabilization of the bacterial membrane dwindles the βlactamase activity, thus sensitizing the bacteria toward β-lactam antibiotics, however, the exact mechanism governing the observed synergy is yet to be discovered. 39,40 The treatment of MRSA infection is further complicated by the formation of biofilm, which restricts the activity of drugs, elongating the treatment process and thus the economic burden on the patients.…”

“…As seen from Table 1, the MIC of Ana-9 was found to be 5.68 μM, while for Ana-10, the MIC value was found to be 2.84 μM against all three tested ATCC strains, which falls in line with our previous observation. 26 Further, the MIC of Ana-9 was found to vary between 2.84 μM to 11.36 μM against HA-MRSA, whereas, for Ana-10, the MIC varied between 1.42 μM to 5.68 μM. Importantly, in addition to the resistance toward oxacillin, 90% of the HA-MRSA isolates tested were found to be resistant to ciprofloxacin.…”

“…For Ana-9, the MBIC was found to be 22.72 μM, while for Ana-10, the value was 11.36 μM against both ATCC 33591 and ATCC 43300, which is in line with our earlier reported work. 26 For the antibiotic oxacillin, the MBIC was found to be 256 μg/mL, and 64 μg/mL against ATCC 33591 and ATCC 43300, respectively. Additionally, against ATCC 33591, the MBEC for Ana-9, Ana-10, and oxacillin was >45.45 μM, >22.72 μM, and >256 μg/mL, respectively, while against ATCC 43300, the MBEC values were 45.45 μM, 22.72 μM, and 128 μg/mL for Ana-9, Ana-10, and oxacillin, respectively.…”