Development of multimethodological strategies for monitoring biorecognition phenomena of pharmaceutically relevant targets

The comprehension of biorecognition phenomena is pivotal to clarify pathophysiological mechanisms and rationally develop new and more effective drugs. Amongst available analytical approaches to profile the binding partners, mass spectrometry (MS), circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopies have been used to investigate two pharmaceutically relevant targets: human serum albumin (HSA) and human cholinesterases (ChEs). Because of the role of HSA in several biological functions and its use in clinical practice as biological drug, it represents an important object of research. In this scenario, two studies were conducted. In the first, the binding capacity of pharmaceutical-grade HSA for infusion was studied by implementing a CD spectroscopy-based assay. A clear impairment of the binding capacity caused by the stabilizers sodium octanoate and N-acetyltryptophan, along with the impossibility of removing octanoate by common approaches, were highlighted. In the second study, SPR- and affinity chromatography-MS-based assays enabled an initial characterization of the interaction between glycated HSA and the receptor for advanced glycation end products (RAGE), providing further insights into the biorecognition phenomenon and laying the groundwork for subsequent studies on AGEs as more representative players in diabetes. On the other hand, ChE enzymes are widely studied molecular targets in drug discovery for Alzheimer’s disease. Their inhibition still represents the main strategy to temporally counteract cognitive impairment and ameliorate patients’ quality of life. In this context, in solution functional assays were used for the identification of new potential inhibitors while a tailored SPR-based assay was developed to complement classic inhibition studies, helping the prioritization of favorite chemical scaffolds by providing affinity and kinetic parameters, including residence time. Overall, the tailored analytical strategies herein reported allowed to elucidate key biorecognition events, providing pivotal information which can help understanding pathological events as well as favoring the drug discovery process

Prospective serological evaluation of anti SARS-CoV-2 IgG and anti S1-RBD antibodies in a community outbreak

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Discovery of Dual Aβ/Tau Inhibitors and Evaluation of Their Therapeutic Effect on a Drosophila Model of Alzheimer's Disease

Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood−brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 μM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ42 expressing flies and generating a better outcome than doxycycline (50 μM). Moreover, 22 proved to be able to decrease Aβ42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in A

ICAROS (Italian survey on CardiAc RehabilitatiOn and Secondary prevention after cardiac revascularization): temporary report of the first prospective, longitudinal registry of the cardiac rehabilitation network GICR/IACPR

The Italian survey on CardiAc RehabilitatiOn and Secondary prevention after cardiac revascularization (ICAROS) was a multicenter, prospective, longitudinal survey carried out by the Italian Association on Cardiovascular Prevention and Rehabilitation (GICR/IACPR) in patients on completion of a CR program after coronary artery by pass grafting (CABG) and percutaneous coronary intervention (PCI). The aim was to evaluate in the short and medium term: i) the cardioprotective drug prescription, modification and adherence; ii) the achievement and maintenance of recommended lifestyle targets and risk factor control and their association with cardiovascular events; iii) the predictors of non-adherence to therapy and lifestyle recommendations. The ICAROS results offers a portrait of the “real world” of clinical practice concerning patients after CABG and PCI, and stresses the need to improve secondary prevention care after the index event: many patients after revascularization leave the acute wards without an optimal prescription of preventive medication but the prescription of cardiopreventive drugs and risk factors control is excellent after completion of a CR program. Following CR, the maintenance of evidence-based drugs and lifestyle adherence at one year is fairly good as far as the target goals of secondary prevention are concerned, but to investigate the influence of CR on long term outcome longer term studies are required. Last, but not least, ICAROS shows that some characteristics (PCI as index event, living alone, poor eating habits or smoking in young age, and old age, in particular with comorbidities) may identify patients with poor behavioral modification in the medium term follow-up and in these patients further support may be warranted. In conclusion, participation in CR results in excellent treatment after revascularization, as well as a good lifestyle and medication adherence at 1 year and provides further confirmation of the the benefit of secondary prevention

Combination of human acetylcholinesterase and serum albumin sensing surfaces as highly informative analytical tool for inhibitor screening

In the continuous research for potential drug lead candidates, the availability of highly informative screening methodologies may constitute a decisive element in the selection of best-in-class compounds. In the present study, a surface plasmon resonance (SPR)-based assay was developed and employed to investigate interactions between human recombinant AChE (hAChE) and four known ligands: galantamine, tacrine, donepezil and edrophonium. To this aim, a sensor chip was functionalized with hAChE using mild immobilization conditions to best preserve enzyme integrity. Binding affinities and, for the first time, kinetic rate constants for all drug–hAChE complexes formation/disruption were determined. Inhibitors were classified in two groups: slow-reversible and fast-reversible binders according to respective target residence time. Combining data obtained on drug–target residence time with data obtained on serum albumin binding levels, a good correlation with potency, plasma protein binding in vivo, and administration regimen was found. The outcomes of this work demonstrated that the developed SPR-based assay is suitable for the screening, the binding affinity ranking and the kinetic evaluation of hAChE inhibitors. The method proposed ensures a simpler and cost-effective assay to quantify kinetic rate constants for inhibitor–hAChE interaction as compared with other proposed and published methods. Eventually, the determination of residence time in combination with preliminary ADME studies might constitute a better tool to predict in vivo behaviour, a key information for the research of new potential drug candidates

Immobilized enzyme-based analytical tools in the -omics era: recent advances

Protein analysis is a field under rapid development mainly thanks to technological advances which have granted miniaturization of analytical devices, automation and higher detection sensitivity. The interest in the field has paralleled the expansion of the -omics era, laying down the bases for the current applications in proteomics and glycomics. Advances in protein sample transformation prior to analysis have led to reduction of sample consumption and contamination, enhancing throughput. Within this context, and thanks to the availability of new high performing materials and technologies, increasingly more efficient and miniaturized enzyme-based analytical tools have been proposed to overcome shortcomings encountered in the in-solution enzymatic reactions (protein digestion and protein deglycosylation, for proteomics and glycomics, respectively). In this context, immobilized enzyme reactors (IMERs) and IMER-based platforms have been developed as promising approaches toward automation and higher analysis throughput. The scenario is in continuous development as underlined by thirty-four papers published in the last five years. This review encompasses recent advances in the design and operational set-ups of IMERs purposely developed for the analysis of proteins and glycoproteins. Recently developed dual IMERs, which integrate more than one processing step into a single IMER, and analytical platforms exploiting tandem IMERs are also reviewed and commented