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A multidisciplinary approach to the design of novel inhibitors for KPC-2
No full textBackground. The emergence and dissemination of multi drug resistant (MDR) Gram-negative pathogens resistant to all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella Pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last resort carbapenems. [1-2]
Materials/methods. Four boronic acid derivatives were designed and tested in vitro vs KPC-2.[3] In biological assays their ability to synergize last generation antibiotics was evaluated. X-ray crystallography was applied to confirm binding orientation and new compounds ability to reach consensus-binding sites in carbapenemases (Figure 1).
Results. For the most actives active compounds nanomolar affinity was achieved. The best inhibitor has nanomolar affinity for the enzyme, a ligand efficiency of 0.78 kcal mol–1 and a molecular weight of 158 Da validating it as lead-like molecule. In biological assays against Escherichia coli overexpressing KPC-2 new derivatives restored susceptibility to cefotaxime, aztreonam and last resort carbapenems. Two crystallographic binary complexes of the best inhibitors binding KPC-2 were obtained at high resolution.
Conclusion. We investigate the molecular recognition requirements in KPC-2 active site by boronic acid derivatives. Kinetic descriptions of slow binding, time dependent inhibition and interactions geometries in KPC-2 were fully investigated.
This study will guide further lead optimization and development of more effective KPC-2 inhibitors.
Figure 1
References
[1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print).
[2] Tondi, D.; Cross, S.; Venturelli, A.; Costi, MP; Cruciani, G.; Spyrakis, F. Current Drug Targets 2016 17, no. 9 (2016)
[3] Tondi,D.; Venturelli,A.; Bonnet,R.; Pozzi, C.; Shoichet, BK.; Costi, M.P. JMC. 2014. 57 (12), pp 5449–5458
New weapons against antimicrobial resistance: Targeting SOS response to recover b actericidal activity of antibiotics
No full textDrug Resistant Bacteria represent a global emergency, limiting the effective treatment of bacterial infections.The development of novel strategies fighting bacterial infections is strongly desirable. One such approach is a means of prolonging the lifespan of current antibiotics, namely, the development of antibiotic adjuvants, i.e. compounds that do not themselves kill bacteria but instead enhance the effect of an antibiotic.
Adjuvants have potential application in a number of situations, with the most obvious being the case of formerly susceptible bacteria that have acquired resistance. Additionally, the relative infancy of the adjuvant approach means that there are likely a greater number of both undiscovered targets and previously unidentified chemical scaffolds
Designing novel inhibitors for Metallo Beta-lactamases
No full textThe extensive use of eta-lactam antibiotics has created major resistance problems leading to increased morbidity, mortality and health-care costs. Resistance is most often mediated by eta-lactamases (BLs), which have emerged in both Gram-positive and Gram-negative bacteria [1,2]. Medicinal chemists have introduced eta-lactam-based molecules that inhibit or are stable to their action. These molecules are themselves eta-lactams, making it easier for bacteria to respond by adapting previously evolved mechanisms. Many bacteria are now resistant to these anti-resistance compounds. Thus there is a growing need for new broad-spectrum eta-lactamases inhibitors in general and especially against Metallo BLs (MBLs)[3,4]. Their substrate promiscuity, their resistance to available drugs, the easiness of variants appearance and transferability make MBLs the most worrisome BLs [2].
Focusing on the de novo, non beta-lactam like derivatives we identified, through a structure-based in silico screening of commercially available library using FLAP, several promising candidates active against class B beta-lactamases (MBL). The binding affinities of the high scoring hits were measured in vitro revealing, for some of them, low micromolar affinity towards BLs.
To investigate the potential of these compounds to reverse antibiotic resistance, we are undertaking antimicrobial activity studies in bacterial cell culture. The ability of novel compounds to synergize antibiotics against pathogenic resistant bacteria, as well as their ability to evade those mechanisms normally involved in resistance to eta-lactam-based inhibitors are now under evaluation. Moreover, since our inhibitors are novel, non eta-lactam based, we expect them to do not up-regulate eta-lactamase expression in cell culture.
X-ray crystallography studies are now in progress to confirm our docking prediction and to deeply investigate the structural requirement necessary for proficuous hit to lead generation.
Keywords: resistance, SBDD, in silico screening, enzyme inhibition, antimicrobial activity, x-ray crystallograph
Structure based drug design: the discovery of novel inhibitors active against New Delhi Metallo-β-lactamase-1
No full textBackground. The emergence of bacterial strains resistant to available antibiotic armamentarium is nowadays a pressing issue in clinical therapy. Among the several mechanisms of bacterial resistance, the rapid evolution and spread of carbapenemases, β-lactamases (BLs) with versatile hydrolytic capacities able to inactivate last resort carbapenems, represents a menace in treating highly resistant infections. [1-3]
Carbapenem-resistant Enterobacteriaceae (CREs), reported with increased frequency, are progressively spreading throughout the World thus leaving no effective antibiotics. [1-3] In particular CREs overexpressing Metallo beta-lactamases (MBLs; i.e. NDM-1, VIM-2, IMP-1) are high-priority target pathogens for the development of novel antibacterials.
Our studies focused on NDM-1: its substrate promiscuity, its resistance to available drugs, the easiness of variants appearance and transferability make NDM-1 one of the most worrisome BLs. [2] Moreover, respect to serine β-lactamases, no inhibitors for any MBLs are currently available in therapy.
Materials/methods. Looking for new potential MBLs inhibitors we performed a structure-based in silico screening of commercially available library using FLAP, and identified several non β-lactam derivatives as promising candidates active against our target New Delhi metallo-beta-lactamase-1 NDM-1. Candidates were validated in vitro and investigated for their mechanism of inhibition.
Results. The binding affinities of the highest scoring hits revealed, for several of them, micromolar inhibitory activity towards NDM-1. Among molecules selected for targeting NDM-1 few demonstrated an activity comparable to that provided by known inhibitors (Ki for the best-selected inhibitor equal to 0.72 μM; ). The identified inhibitors all share common non-covalent, competitive inhibition mechanism vs NDM-1.
Conclusion. For the best ones, studies for improving their affinity and to investigate their potential to synergize beta-lactam antibiotics are ongoing. X-ray crystallography studies are now in progress to confirm our docking prediction and to deeply investigate the structural requirement necessary for proficuous hit to lead generation.
Figure 1
References
[1] Jean-Marie Frère, Eric Sauvage and Frédéric Kerff. “From “An enzyme able to destroy penicillin » to carbapenemases: 70 years of beta-lactamase misbehavior” Current Drug Targets, (2016). Volume 16. (E-pub ahead of print).
[2] Robert A. Bonomo. “New Delhi Metallo-β-Lactamase and Multidrug Resistance: A Global SOS?”Clin Infect Dis. (2011) 52 (4): 485-487
[3] Donatella Tondi, Alberto Venturelli, Richard Bonnet, Cecilia Pozzi, Brian K Shoichet and Maria Paola Costi. “Targeting Serine Beta lactamases with a novel broad spectrum boronic acid”. Journal Medicinal Chemistry. (2014
Structurally Related Liposomes Containing N-Oxide Surfactants: Physicochemical Properties and Evaluation of Antimicrobial Activity in Combination with Therapeutically Available Antibiotics
Full text linkAlthough liposomes are largely investigated as drug delivery systems, they can also exert a pharmacological activity if devoid of an active principle as a function of their composition. Specifically, charged liposomes can electrostatically interact with bacterial cells and, in some cases, induce bacterial cell death. Moreover, they also show a high affinity toward bacterial biofilms. We investigated the physicochemical and antimicrobial properties of liposomes formulated with a natural phospholipid and four synthetic L-prolinol-derived surfactants at 9/1 and 8/2 molar ratios. The synthetic components differ in the nature of the polar headgroup (quaternary ammonium salt or N-oxide) and/or the length of the alkyl chain (14 or 16 methylenes). These differences allowed us to investigate the effect of the molecular structure of liposome components on the properties of the aggregates and their ability to interact with bacterial cells. The antimicrobial properties of the different formulations were assessed against Gram-negative and Gram-positive bacteria and fungi. Drug-drug interactions with four classes of available clinical antibiotics were evaluated against Staphylococcus spp. The target of each class of antibiotics plays a pivotal role in exerting a synergistic effect. Our results highlight that the liposomal formulations with an N-oxide moiety are required for the antibacterial activity against Gram-positive bacteria. In particular, we observed a synergism between oxacillin and liposomes containing 20 molar percentage of N-oxide surfactants on Staphylococcus haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus. In the case of liposomes containing 20 molar percentage of the N-oxide surfactant with 14 carbon atoms in the alkyl chain for S. epidermidis, the minimum inhibitory concentration was 0.125 mu g/mL, well below the breakpoint value of the antibiotic
Protolichesterinic acid enhances doxorubicin-induced apoptosis in HeLa cells in vitro
No full textAim The aim of this study was to investigate the effect of protolichesterinic acid, a lichen secondary metabolite, on anti-proliferative activity of doxorubicin in three human cancer cell lines, HeLa, SH-SY5Y and K562 cells. Main methods The data obtained from MTT assays, performed on cells treated with protolichesterinic acid and doxorubicin alone and in combination, were analysed by the median-effect method as proposed by Chou and Talalay and the Bliss independence model. Apoptosis rate was evaluated by fluorescence microscopy, caspase-3, 8 and 9 activities were detected by spectrofluorimetric analysis and protein expression of Bim, Bid, Bax and Mcl-2 was analysed by Western blotting. The interaction of protolichesterinic acid with thioesterase domain of human fatty acid synthase (hFAS) was investigated by a molecular docking study. Key findings The in vitro activity of doxorubicin against HeLa cancer cell line, but not against SH-SY5Y and K562 cells, was synergically increased by protolichesterinic acid. The increased cytotoxicity caused by protolichesterinic acid in HeLa cells was due to a pro-apoptotic effect and was associated to caspase-3, 8 and 9 activation. The simultaneous treatment for 24 h with protolichesterinic acid plus doxorubicin caused an increase of Bim protein expression and the appearance of cleaved form of Bid protein. The molecular modelling analysis showed that protolichesterinic acid seemed to behave as a competitive inhibitor of hFAS. Significance These results suggest that protolichesterinic acid could be envisaged as an useful tool against certain types of tumor cells in combination with anticancer drugs
First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase
No full textThe worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase
Curcuminoids-loaded liposomes: influence of lipid composition on their physicochemical properties and efficacy as delivery systems
Full text linkAn extract of Curcuma longa containing mainly curcumin was included in liposomes formulated with phospholipids differing for the length of the acyl chains, the charge of the polar headgroup or the degree of unsaturation in the presence and in the absence of cholesterol. In fact, even if it is well known that liposomal curcuminoids are more stable and can be solubilized in aqueous media, a systematic approach that correlates the molecular structure of liposomes components to the physicochemical and biological behavior of the vescicles including curcumin and its derivatives is not described. Size and zeta potential of liposomes, the location and the antioxidant activity of the solute in the bilayer and its effect on lipid packing were evaluated and correlated to the pharmacological activity of liposomal extract against a methicillin susceptible Staphylococcus Aureus strain ATCC 29213. The relation between the physicochemical and biological features of formulations points out that the rigidity of the bilayer and liposomes charge are crucial parameters in the interactions of liposomes with the biological environment. In particular, the synergism between curcumin antibacterial properties and the positive liposomes surface brings to a significant reduction of the minimal inhibitory concentration of the liposomal drug with respect to the one of the free extract thus enlarging the prospectives of its pharmacological exploitation. © 2020 Elsevier B.V
Influence of Lipid Composition on Physicochemical and Antibacterial Properties of Vancomycin-Loaded Nanoscale Liposomes
Full text linkThe bacterial resistance against antibiotics has reached an alarming level, leading to an increase in morbidity and mortality. One of the possible approaches to tackle this problem of major concern for the global community is to renovate “old” antibiotics to make pathogens susceptible to their pharmacological activity. Vancomycin (VAN) is a glycopeptide used for the treatment of Gram-positive bacterial infections such as the ones caused by methicillin-resistant Staphylococcus aureus. Nevertheless, some strains of this bacterium developed high-level VAN resistance. To revert VAN resistance and decrease its side effects, we included this antibiotic in different liposomal formulations. Nanoscaled liposomes were formulated with pure phospholipids differing in the presence of unsaturation and/or in the length of the acyl chains. DPPC liposomes were also tested in a mixture with cholesterol hemisuccinate (at 40 molar percentage) and/or a cationic saturated lipid (at 30 molar percentage) to make them pH-responsible and/or positively charged. Two techniques for liposome preparation, thin film hydration and reverse phase evaporation, were employed and compared. The key element of this investigation is a systematic and rational variation of liposomes composition and VAN loading technique to modulate the physicochemical properties of the formulations. The availability of a “revised” broad-spectrum VAN could increase its therapeutic options in the case of bacterial infections and, at the same time, limit the failure of the treatment due to the beta-lactam resistance in bacteria
Salvianolic Acid B Strikes Back: New Evidence in the Modulation of Expression and Activity of Matrix Metalloproteinase 9 in MDA-MB-231 Human Breast Cancer Cells
Full text linkSalvianolic acid B (SalB) is a bioactive compound from Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9
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