Evaluation of inhibitory action of novel non Œ≤-lactam inhibitor against Klebsiella pneumoniae carbapenemase (KPC-2)
Authors: Asad U Khan
Mohammad Faheem
Arbab Khan
Mohd Danishuddin
Journal: PloS one (2014)
Discipline: Multidisciplinary
Keywords: Œ≤-lactamase inhibitors; carbapenemase KPC-2; novel inhibitors; antibacterial activity; MIC values
Affiliations: Medical Microbiology and Molecular Biology Lab, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.

Abstract:  The use of three classical Œ≤-lactamase inhibitors (Clavulanic acid, tazobactam and sulbactam) in combination with Œ≤-lactam antibiotics is presently the mainstay of antibiotic therapy against Gram-negative bacterial infections. However these inhibitors are unable to inhibit carbapenemase KPC-2 effectively. They being Œ≤-lactam derivatives behave as substrates for this enzyme instead of inactivating it. We have initiated our study to check the in vitro inhibition activity of the two novel screened inhibitors (ZINC01807204 and ZINC02318494) in combination with carbapenems against KPC-2 expressing bacterial strain and their effect on purified enzyme KPC-2. The MIC values of meropenem and ertapenem showed maximum reduction (8 folds) in combination with screened compounds (ZINC01807204 and ZINC02318494). CLSM images also depicted their strong antibacterial activity in comparison to conventional Œ≤-lactamase inhibitors. Moreover no toxic effect has been shown on HeLa cell line. Though the IC50 value of ZINC01807204 was high (200 ¬µM), it exhibited fairly good affinity for KPC-2 (Ki = 43.82 ¬µM). With promising results this study identifies ZINC01807204 as a lead molecule for further optimization and development of more potent non Œ≤-lactam inhibitors against KPC-2. 

Retracted: No

PubPeer comments:
 The cloning strategy and purification of KPC-2 used in this study is rather controversial. Based on the primers and cloning vector used (pQE-2), the authors have expressed full length native KPC-2 (~32.33 kDa) with a N-terminal hisTAG, just upstream of natural peptide leader targeting the protein in periplasm.  The authors claim to have purified KPC-2 using Histidine affinity TAG from whole-cell lysates. **How is that possible if the His-TAG gets away with the cleaved off peptide leader upon periplasmic translocation?** Even assuming that they purified only a fraction of his-tagged full length KPC-2 that was not translocated for an inefficient export process (this could happen when translocation machinery is overloaded or possibly due the modification of peptide leader), **then why does the purified protein have a molecular weight (~29 kDa based on Fig. S1) corresponding to the truncated KPC-2 (i.e. devoid of leader sequence)?**  Purification of KPC-2. SDS-PAGE of induced KPC-2 protein in supernatant and pellet of bacterial cells after sonication in lane 1 and 2 respectively. Lane 3, 4 and 5 represents purified KPC-2 protein in subsequent elutions. The single band represents molecular mass of approximately 29 kDa. Lane M is protein marker. , 
 We agree with the comments. The cloning strategy and purification of KPC-2 used in this study Based on the primers and cloning vector used (pQE-2) with a N-terminal hisTAG, and found that after purification of the KPC-2 protein size 29 kD was estimated only by the protein marker. We donu2019t claim size of the protein without going to other experiment like LCMS. In this study We didnu2019t do experiment related to size of the protien. We agree all point of the commenter. But what ever priten we got expressed and purified first activity check. ,