Treatment of infectious diseases is difficult due to increased bacterial resistance and also is a significant global health concern.²⁴ Beta-lactamases have developed mutations that could exhibit resistance to inhibitors.⁸ Despite the effectiveness of some combination regimens, beta-lactamase enzymes mutate exhibiting resistance to the inhibitors and consequently, emphasizing the requirement of newer drugs or a newer combination regime.

The unique loop structure known as the omega loop is a non-regular structural motif resembling the upper-case Greek letter omega (Ω), is present in all serine beta-lactamases and is crucial for maintaining the stability between the enzyme and substrate. Specifically, it has been demonstrated that the conserved Ω-loop of class A beta-lactamases is essential to the hydrolytic process during beta-lactam hydrolysis. The omega loop (Ω-loop), a conserved non-active site structural domain seen in class A serine β-lactamases (SBLs), is thought to contain a glutamic acid residue that directly contributes to the hydrolysis of β-lactam antibiotics by supplying a water molecule during catalysis. Hence, targeting the β-lactamases’ Ω-loop could increase the potency of β-lactam antibiotics and ultimately decrease β-lactam resistance. Thus, In this study, the cyclic peptides were screened to target against the conserved Ω-loop of class A beta-lactamases and thus subsequently the identified hit compounds may function as inhibitors of β-lactamase, lowering the minimum inhibitory concentrations and profoundly altering the resistance profile of β-lactams in bacteria.

Among the peptides which showed the highest docking score Elafin, Cinnamycin, Duramycin interacted with Lys 73 of the a domain of catalytic residues of TEM-1 Beta-lactamases whereas Taromycin A, Gassericin A interacted with Lys 234 of the b domain. However, none of the peptides showed any interaction with the other residues of the catalytic site of the SDN-loop amino acids (Asn132 & Ser130) and Ω-loop residues (Glu166 & Asn170).

Lys 73 residue in the Ω-loop of TEM-type beta-lactamase in its apo form interacts with Glu166. The residues Asn170 and Lys 73 of the Ω-loop formed hydrogen bond in the active site when Glu166 loses a proton and the distributed charged residues of active sites have been altered during the binding of antibiotics to the enzyme. Specifically, Lys73 residue deprotonates and attacks Ser70 nucleophilically, causing it to deprotonate as well. An acyl-enzyme covalent complex is produced as a result of this interaction of the Beta-lactam ring with antibiotic’s carbonyl carbon atom. Hence the compounds Elafin, Cinnamycin, and Duramycin interacting with Lys 73 of TEM-1 beta-lactamases may aid in preventing resistance and the hydrolysis process.¹⁷

All serine beta-lactamases of classes A, C, and D have an expanded second loop referred to as the Ω-loop. The Ω-loop of TEM-type β-Lactamases is situated at the base of the active region of the enzyme and consists of 16 residues (Arg164–Asp179). Glu166 is an essential component in the hydrolysis of β-lactams and is substantially conserved in class A β-Lactamases. However, none of the peptides interacted with Glu 166 in our study.¹⁷

Asp 4 of Guentherin A interacted with the Asn 170 residue located at the bottom of the enzymatic active site of TEM-type Ω-loop bLs which is comprised of 16 aminoacid residues from Arg164 to Asp179. Met 14 of the peptide interacted with the Chrombacin Ala 172 at the hydrogen bond distance of 2.70, and Agr 18 of Gassericin A interacted with the Pro 167 at 2.83. Notably, none of the compounds tested reacted with the highly prevalent residue Glu166 of class A, which is important for the hydrolysis of β-lactams.²⁵

The N-terminal portion for the omega loop (Arg164–Asn170 residues) in TEM-type bLs has a stiff conformation, while Arg 164 is involved in maintaining bottleneck loop structure. The proximity of these residues to one another can be attributed to a-helix formation by the Pro167–Asn170 residues.

Before the catalytically active orientations form, the Peptidyl-proline bond (Glu166–Pro167) must be formed in trans configuration and the hydrogen bonding complex occurs with Glu166, Asn170 and water molecule.¹⁷ However, the disturbances at these specific sites were not observed in our interaction analysis.

Mutations in the loop destabilize the conformation due to the rapid exchange of water molecules close to the Ω-loop, thus increasing the catalytic effectiveness of ceftazidime and cefotaxime hydrolysis, in specific, the substitution Arg164Ser maintains a hydrogen bond between Ser164 and Asp179, which further stabilize the omega loop conformation and has been observed commonly amongst clinical strains producing TEM-type β-Lactamases.²⁶

Hence, the design and identification of new molecules that could inhibit this process of inactivation are required to overcome this resistance, and hence the screened peptides in this study provide a clear insight into the interaction with the active site and also on their conformation. According to CAPRI (Critical Assessment of Predicted Interactions) experiments, HADDOCK and ClusPro are found to be the best automated protein docking systems. HADDOCK uses Fast Fourier Transforms (FFTs)-based rigid-body docking techniques and as a part of the preliminary investigation in this study, the cyclic peptides were subjected to the class A Beta-lactamases Omega loop, and hence in futuristic studies , the lead compound could be further validated by in vitro antibacterial studies against the resistant strains.^27-29

Substantial reports exist on the antimicrobial activity of reported peptides. Gassericin A bacteriocin produced by Lactobacillus gasseri LA39 has shown antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus.³⁰ Likewise, the 19-amino acid tetracyclic lanthipeptide Duramycin by streptomycetes and  Brevinin-1 ,skin peptide from  H. rugulosus exhibited antimicrobial activities.

Cinnamycin (19-residue tetracyclic peptide) of the lantibiotic family possesses the thioether amino acids meso-lanthionine and (2S,3S,6R)-3-methyllanthionine and are formed by cross-linking serine or threonine with cysteine, and has the potential to locate and rupture PE-containing membranes, including those found in bacteria and cancer cells, due to its specific binding to PE lipids.³¹

Elafin is an antimicrobial peptide that has exhibited antibacterial activity against  Staphylococcus aureus and Pseudomonas aeruginosa, and likewise, Japonicin-1 has demonstrated effective antimicrobial activity against Staphylococcus aureus and MRSA. Lactocyclicin Q has a cyclic structure with a 19-residue tetracyclic peptide bound at N and C termini and showed antimicrobial activity against Lactococcus and Enterococcus. Taromycins A and B showed calcium ion-dependent broad-spectrum antibacterial activity against MRSA and E. faecium.³²

Therefore, our research provides more details on how these specific peptides interact with TEM-Bl, which could aid in the development of efficient medications to combat bacterial strains that are resistant to current beta-lactam antibiotics and also for a combinational regime.