Half maximal inhibitory concentration values for ZINC01807204 and other β-lactamase inhibitors determined after 5 minute of incubation with KPC-2.

<p><i>IC₅₀</i> Half maximal inhibitory concentration. Shown as arithematic mean (three repetitions on three separate days).</p><p>Half maximal inhibitory concentration values for ZINC01807204 and other β-lactamase inhibitors determined after 5 minute of incubation with KPC-2.</p

Evaluation of Inhibitory Action of Novel Non β-Lactam Inhibitor against <i>Klebsiella pneumoniae</i> Carbapenemase (KPC-2)

<div><p>The use of three classical β-lactamase inhibitors (Clavulanic acid, tazobactam and sulbactam) in combination with β-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 β-lactam derivatives behave as substrates for this enzyme instead of inactivating it. We have initiated our study to check the <i>in vitro</i> 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 β-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 (K_i = 43.82 µM). With promising results this study identifies ZINC01807204 as a lead molecule for further optimization and development of more potent non β-lactam inhibitors against KPC-2.</p></div

Determination of IC₅₀ values for different inhibitors.

<p>The residual activity of KPC-2 after 5 minutes pre-incubation with varying concentrations of different inhibitors as monitored by the hydrolysis of 100 mM nitrocefin.</p

Effect of different concentrations of screened inhibitors on HeLa cell line for three different time durations (12 hours, 24 hours & 48 hours) determined by MTT assay.

<p>Percent cell survival in the control group was assumed 100%. (A) ZINC01807204 (B) ZIC02318494. n = 3, p<0.05.</p

MICs of carbapenems alone and in combination with inhibitors for <i>E. coli</i> BL21 transformed with recombinant <i>bla</i>_KPC-2 from <i>Klebsiella pneumoniae</i>.

<p>Clavulanic acid, tazobactam, sulabactam, ZINC01807204 and ZINC02318494 were used at fixed concentration of 4 µg/ml. All experiments were repeated atleast thrice.</p><p>MICs of carbapenems alone and in combination with inhibitors for <i>E. coli</i> BL21 transformed with recombinant <i>bla</i>_KPC-2 from <i>Klebsiella pneumoniae</i>.</p

Chemical structure of screened inhibitors (A) ZINC01807204 (B) ZIC02318494.

<p>Chemical structure of screened inhibitors (A) ZINC01807204 (B) ZIC02318494.</p

Molecular docking of various inhibitors at the active site of CTX-M-15.

<p>Panel (A) shows molecular docking of clavulanic acid (red), sulbactam (green), tazobactam (yellow) and screened compound ZINC03787097 (blue) at the active site of CTX-M-15 from clinical strain of <i>Enterobacter cloacae</i> (EC-15). Panel (B) represents close view of interacting amino acids in which dashed lines represent hydrogen bonds. Ambler positions of amino acids are Ser-73 (Ser-42), Tyr-108 (Tyr-77), Ser-133 (Ser-102), Asn-135 (Asn-104), Asn-173 (Asn-141), Gly-239 (Gly-208), Ser-240 (Ser-209), Thr-218 (Thr-188).</p

CLSM images of <i>E. coli</i> BL21 transformants harbouring <i>bla</i>_KPC-2 4 hours after treatment with sub-MIC concentration of (B) Meropenem (C) Meropenem + tazobactam (D) Meropenem + ZINC01807204 (E) Meropenem + ZIC02318494 (A) Control, no treatment.

<p>CLSM images of <i>E. coli</i> BL21 transformants harbouring <i>bla</i>_KPC-2 4 hours after treatment with sub-MIC concentration of (B) Meropenem (C) Meropenem + tazobactam (D) Meropenem + ZINC01807204 (E) Meropenem + ZIC02318494 (A) Control, no treatment.</p

Biochemical Characterization of CTX-M-15 from <em>Enterobacter cloacae</em> and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

<div><p>The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of <i>bla</i>_CTX-M-15 gene in different clinical strains of <i>Enterobacteriaceae</i> from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on <i>E. coli</i> DH5α transformed with <i>bla</i>_CTX-M-15 gene that was cloned from <i>Enterobacter cloacae</i> (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC₅₀ value (6 nM), high affinity (<i>K</i>_i = 0.017 µM) and better acylation efficiency (<i>k</i>₊₂/<i>K</i>′ = 0.44 µM⁻¹s⁻¹). It forms an acyl-enzyme covalent complex, which is quite stable (<i>k</i>₊₃ = 0.0057 s⁻¹). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC₅₀ (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (<i>K</i>_i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.</p> </div

Concentration of the inhibitors required to reduce the enzyme activity by 50%.

<p>Concentration of the inhibitors required to reduce the enzyme activity by 50%.</p