Can We Exploit β-Lactamases Intrinsic Dynamics for Designing More Effective Inhibitors?

β-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics

Structure-based discovery and in-parallel optimization of novelcompetitive inhibitors of thymidylate synthase

AbstractBackground:The substrate sites of enzymes are attractive targets for structurebased inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging.Results:In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and clocking studies led to a derivative of one of these, dansyltyrosine (Ki 65 μM. Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 μM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes.Conclusions:TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged

Enhancing mechanical and surface properties of eucalyptus wood

Eucalyptus is one of the most fast-growing trees. Therefore, in the last decades it has been extensively planted and harvested so that nowadays Eucalyptus is one of the most popular trees of the planet. There are many genres of this plant and they are often treated as a large bunch of the same timber characterized by moderate mechanical and surface properties which hinder their usage for any sight application (e.g. flooring, cladding, ceiling). In this study four species of Eucalyptus: E. grandis, E. dunnii, E. cloeziana and E. tereticornis were undergone to densification through hydro-thermo-mechanical treatment (HTM) first and then to oil heat-treatment (OHT) in order to improve their mechanical properties and hydrophobicity. It was observed that low density species (E. grandis) reaches higher compression degrees while heavier species (E. tereticornis) reach densities over 800 kg/m³; however, HTM decrease the variability of the properties. Treatments at higher temperature (160 °C) involves higher compression degree, lower set-recovery and higher surface hydrophobization, but also weaker mechanical properties. The hot oil post- treatment helps to contain the springback effect and to reduce the wettability of each specimen. Densified samples present similar surface hardness. The tailored application of the two treatments improves the properties of every Eucalyptus which can gain market also for nobler end-usages

Faulting of a turbidite sandstone-siltstone successions: the case study of the Macigno Formation, Tuscany, Italy

Faults in siliciclastic rocks are characterized by a great variability of fault zone architecture and relative permeability properties. This is because siliciclastic rocks (i.e turbidites) are often represented by alternating beds of various thickness and grain size forming a succession of strata with contrasting mechanical properties. For example, the presence of sandstone and clay-rich layers is responsible for the simultaneous occurrence of brittle and ductile deformation, known as “clay smear structures”. Moreover, numerous studies have identified grain size as one of the main influencing factors for fault nucleation processes and fracture intensity in the damage zone. In this work, we present the results of field and laboratory analyses performed on the Macigno Formation cropping out along the coast of western Tuscany. Here, the Macigno Formation is represented by Late Oligocene foredeep siliciclastic succession dominated by turbiditic sandstones with minor siltstones, mudstones, marls and shales. Thin section and 3D analyses, performed by X-ray Synchrotron tomography, allowed us to characterize the grain size and grain and cement composition of studied rocks. Grain size varies from channelized fine-grained sandstones to granule-conglomerates beds (0.006 mm to 4 mm) alternating with heterolithic levee strata of siltstones to fine-grained sandstones (0.0035-0.008 mm). The lithic components consist of metamorphic rocks by 70-80%, magmatic rocks by 15-20% and sedimentary rocks by 5-15%. The turbidite beds are normally well-cemented (by quartz and calcite) and heavily faulted and fractured. Investigated faults show dip-, oblique- and and strike-slip motion and their displacement range from 10s of centimetres to 10s of metres. We documented how both the grain size and the mechanical properties of the alternating beds strongly control the fault zone architecture, in particular in terms of damage zone thickness and fracture frequency. The fault rock types (i.e. breccia vs. gauge) are strictly related to the amount of displacement as well as to the grain size and the cementation of the sandstone. Furthermore, the development of clay smear structures are enhanced by the presence of interbedded thin clay-rich layers

Fully Biobased Polyhydroxyalkanoate/Tannin Films as Multifunctional Materials for Smart Food Packaging Applications

Fully biobased and biodegradable materials have attracteda growinginterest in the food packaging sector as they can help to reduce thenegative impact of fossil-based plastics on the environment. Moreover,the addition of functionalities to these materials by introducingactive molecules has become an essential requirement to create modernpackaging able to extend food's shelf-life while informingthe consumer about food quality and freshness. In this study, we presentan innovative bioplastic formulation for food packaging based on poly-(hydroxybutyrate-co-valerate) (PHBV) and tannins as multifunctional additives.As a proof of concept, PHBV/tannin films were prepared by solventcasting, increasing the tannin content from 1 to 10 per hundred ofresin (phr). Formic acid was used to reach a homogeneous distributionof the hydrophilic tannins into hydrophobic PHBV, which is remarkablychallenging by using other solvents. Thanks to their well-known properties,the effect of tannins on the antioxidant, UV protection, and gas barrierproperties of PHBV was evaluated. Samples containing 5 phr bioadditiverevealed the best combination of these properties, also maintaininggood transparency. Differential scanning calorimetry (DSC) investigationsrevealed that films are suitable for application from the fridge topotentially high temperatures for food heating (up to 200 degrees C).Tensile tests have also shown that Young's modulus (900-1030MPa) and tensile strength (20 MPa) are comparable with those of thecommon polymers and biopolymers for packaging. Besides the improvementof the PHBV properties for extending food's shelf-life, itwas also observed that PHBV/tannin could colorimetrically detect ammoniavapors, thus making this material potentially applicable as a smartindicator for food spoilage (e.g., detection of fish degradation).The presented outcomes suggest that tannins can add multifunctionalproperties to a polymeric material, opening up a new strategy to obtainan attractive alternative to petroleum-based plastics for smart foodpackaging applications

Mono- and disubstituted-3,8-diazabicyclo[3.2.1]octane derivatives as analgesics structurally related to epibatidine: synthesis, activity, and modeling.

A series of 3,8-diazabicyclo[3.2.1]octanes substituted either at the 3 position compounds 1) or at the 8 position (compounds 2) by a chlorinated heteroaryl ring were synthesized, as potential analogues of the potent natural analgesic epibatidine. When tested in the hot plate assay, the majority of the compounds showed significant effects, the most interesting being the 3-(6-chloro-3-pyridazinyl)-3,8-diazabicyclo[3.2.1]octane (la). At a subcutaneous dose of 1 mg/kg, 1a induced a significant increase in the pain threshold, its action lasting for about 45 min. 1a also demonstrated good protection at a dose of 5 mg/kg in the mouse abdominal constriction test, while at 20 mg/kg it completely prevented the constrictions in the animals. Administration of naloxone (1 mg/kg ip) did not antagonize its antinociception while mecamylamine (2 mg/kg ip) did, thus suggesting the involvement of the nicotinic system in its action. Binding studies confirmed high affinity for the \u3b1\u3b22 nAChR subtype (K(i) = 4.1\ub10.21 nM). nAChR functional activity studies on three different cell lines showed that 1a was devoid of any activity at the neuromuscular junction. Finally, due to the analogy in their pharmacological profile with that of epibatidine, compounds were compared from a structural and conformational point of view through theoretical calculations and high-field 1H NMR spectroscopy. Results indicate that all of them present one conformation similar to that of epibatidine

Phenylboronic Acids Probing Molecular Recognition against Class A and Class C beta-Lactamases

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, \u392-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine \u392-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect \u392-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with \u392-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors

The Palermo (Sicily) seismic cluster of September 2002 in the seismotectonic framework of the Tyrrhenian Sea-Sicily border area

The northern coast of Sicily and its offshore area represent a hinge zone between a sector of the Tyrrhenian Basin, characterized by the strongest crustal thinning, and the sector of the Sicilian belt which has emerged. This hinge zone is part of a wider W-E trending right-lateral shear zone, which has been affecting the Maghrebian Chain units since the Pliocene. Seismological and structural data have been used to evaluate the seismotectonic behavior of the area investigated here. Seismological analysis was performed on a data set of about 2100 seismic events which occurred between January 1988 and October 2002 in the Southern Tyrrhenian Sea. This paper focuses in particular on a set of data relating to the period from 6th September 2002, including both the main shock and about 540 aftershocks of the Palermo seismic sequence. The distribution of the hypocenters revealed the presence of two main seismogenic zones. The events of the easternmost zone may be related to the Ionian lithospheric slab diving beneath the Calabrian Arc. The seismicity associated with the westernmost zone is closely clustered around a sub-horizontal regression plane contained within the thinned Southern Tyrrhenian crust, hence suggesting that this seismogenic zone is strictly connected to the deformation field active within the hinge zone. On the basis of both structural and seismological data, the brittle deformation pattern is characterized by high-angle faults, mainly represented by transcurrent synthetic right-lateral and antithetic left-lateral systems, producing both restraining/uplifting and releasing/subsiding zones which accommodate strains developing in response to the current stress field (characterized by a maximum axis trending NW-SE) which has been active in the area since the Pliocene. The cluster of the seismic sequence which started with the 6th September 2002's main shock is located within the hinge zone. The distribution of the hypocenters relative to this sequence emphasizes the presence of a high-angle NE-SW-oriented deformation belt within which several shear surfaces are considered to be found sub-parallel to that established for the main shock. The kinematics of all these structures is consistent with a compressive right-lateral focal mechanism