Structural Basis for E. coli Penicillin Binding Protein (PBP) 2 Inhibition, a Platform for Drug Design.

Penicillin-binding proteins (PBPs) are the targets of the ß-lactams, the most successful class of antibiotics ever developed against bacterial infections. Unfortunately, the worldwide and rapid spread of large spectrum ß-lactam resistance genes such as carbapenemases is detrimental to the use of antibiotics in this class. New potent PBP inhibitors are needed, especially compounds that resist ß-lactamase hydrolysis. Here we describe the structure of the E. coli PBP2 in its Apo form and upon its reaction with 2 diazabicyclo derivatives, avibactam and CPD4, a new potent PBP2 inhibitor. Examination of these structures shows that unlike avibactam, CPD4 can perform a hydrophobic stacking on Trp370 in the active site of E. coli PBP2. This result, together with sequence analysis, homology modeling, and SAR, allows us to propose CPD4 as potential starting scaffold to develop molecules active against a broad range of bacterial species at the top of the WHO priority list.

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation.

Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.

BACKGROUND: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction. RESULTS: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket. CONCLUSION: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

Mechanistic studies of the inactivation of TEM-1 and P99 by NXL104, a novel non-beta-lactam beta-lactamase inhibitor.

NXL104 is a potent inhibitor of class A and C serine ß-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 ß-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 ß-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k(2)), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k(2)/K of 3.7 × 10(5) M(-1) s(-1) for TEM-1 and 1 × 10(4) M(-1) s(-1) for P99) and slow decarbamylation. Complete loss of ß-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k(3) value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC(50)s) were evaluated on selected ß-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C ß-lactamases. IC(50)s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the ß-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.

Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases.

NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli, whereas the converse is true of enzymes from Staphylococcus aureus. This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.

The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor.

Calcofluor is an antifungal compound known to induce structural perturbations of the cell wall by interfering with the synthesis of chitin microfibril. Proteins from a stripped plasma membrane fraction were solubilized with the neutral and non-denaturing detergent, the n-dodecyl beta-D-maltoside. Proteins were then resolved using a recently described ion-exchange chromatography (IEC)/lithium dodecyl sulfate (LDS)-PAGE procedure. Nearly 90 proteins were identified and clustered, based on their pI, molecular weight, abundance and/or hydrophobicity. This method was then applied to profile the plasma membrane response to calcofluor. The LDS-PAGE patterns obtained from whole plasma membrane proteins were similar for the non-treated and calcofluor-treated samples. However, IEC/LDS-PAGE analysis revealed subtle changes in the expression of several proteins of low abundance, in response to calcofluor. These proteins include Pil1p and Lsp1p, two sphingolipid long-chain base-responsive inhibitors of protein kinases involved in signaling pathways for cell wall integrity and Rho1p, a small GTPase. It was recently hypothesized that Pil1p and Lsp1p could associate with, and regulate, the plasma membrane beta-1-3-glucan synthase, responsible for the synthesis of another major microfibril for yeast cell wall. Results are discussed with respect to both calcofluor effects on the plasma membrane proteins and the power of the IEC/LDS-PAGE procedure in the search for new potential therapeutics targets.

Drug induced proteome changes in Candida albicans: comparison of the effect of beta(1,3) glucan synthase inhibitors and two triazoles, fluconazole and itraconazole.

The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.