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The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

For the implantation of the PM, iodized contrast medium is rarely used and to recognize the anatomical structures within which one is operating, one must resort to the interpretation of only the radiological images obtained with the use of ionizing radiation. To verify the correct positioning of the leads, it is necessary to have multiple points of view (projections). Confirmation is usually sought with at least two projections that are almost orthogonal to each other. The fluoroscopic visualization shows a two-dimensional antomy typical of radiological imaging and we speak of a cardiac shadow formed by the overlapping of all vascular structures Typical views to visualize correct lead placement require multiple views, usually two. The projections RAO 20-25 ° and LAO 25-30 ° are almost orthogonal. With the first, the cardiac silhouette shows the right ventricle in its maximum longitudinal extension. With the LAO, the left ventricle and the right ventricle are represented paired with a view comparable to the 4-chamber ultrasound imaging Knowledge of radiological anatomy is very important in determining the correct positioning of the leads.

The use of 3D printing in the medical field is widespread especially in the dental clinic as a support to orthodontic / implant therapy. In the training of personnel relating to cardiological diagnostic methods, can it be useful to shorten the learning curve of human anatomy? A light-curing liquid resin 3D printer was used to obtain anatomical models that faithfully reproduce the real anatomy of the individual patient. The starting model was obtained from CT with contrast medium performed on patients. Depending on the diagnostic question, CT may contain complete information on both the arterial circulation and the venous circulation of the heart and the epiaortic vessels. From this information a 3D surface reconstruction is obtained which, once reworked, can be used directly for printing. The model size is limited by the print volume of the printer which may vary depending on the printer model. The model to be printed obtained from CT acquisitions the 3D model will be the representation of the lumen of the vessels and cavities of the heart. The models obtained faithfully represent the anatomical reality of the patient who underwent CT with contrast medium. Actual size is affected by the print volume size of the printer model. In my experience in university teaching, the models obtained with 3D printing have been found to be very useful for understanding the complex spatial configuration of the anatomy of the heart and great vessels

The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.

Cardiomyopathies to be the first cause of death. The patient who suffers from acute heart disease, after clinical evaluation, is subjected to a diagnostic–therapeutic process that involves several professional figures which include the doctor and the ambulance nurse, the doctor and all the emergency room staff who accept the patient and, if the clinical indication is oriented towards a vascular occlusion disease, the doctor and the technical-nursing team of the haemodynamics service activated in urgency. To diagnose with certainty the presence of vascular disease we make use of diagnostic systems that use ionizing radiation; the haemodynamics team thus finds itself operating not only in urgent conditions but also in dangerous conditions due to the presence of ionizing radiation. Nursing and technical staff need additional skills to be able to take on roles from basic care to emergency electric shock resuscitation. The purpose of this work is to illustrate the phases of the activity that stabilize the treatment in the hemodynamics room from the acute phase to the patient.

In the last 50 years, cardiac electrophysiology has undergone rapid technological development which has led to a numerical increase in both patients who have been able to benefit from the therapies of rediscovery and rhythm control, and of the devices. The activity of an electrophysiology and electrostimulation room is based on the intensive use of ionizing radiation even if electrophysiology studies, ablation, and cryoablation techniques have benefited from the support of computerized electroanatomical mapping systems with consequent dose reduction. Over the years, the instruments to be managed inside the room have increased both in complexity and numerically. Starting from the biventricular PM, we go through the implantation of subcutaneous defibrillators up to the transseptal ablations. Patient management requires additional skills that each member of the team must possess. Continuous training and updates are of fundamental importance. The purpose of this work is our experience based on the activity now more 10 years.

The transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are the major downstream effectors in the Hippo pathway and are involved in cancer progression through modulation of the activity of TEAD (transcriptional enhanced associate domain) transcription factors. To exploit the advantages of drug repurposing in the search of new drugs, we developed a similar approach for the identification of new hits interfering with TEAD target gene expression. In our study, a 27-member in-house library was assembled, characterized, and screened for its cancer cell growth inhibition effect. In a secondary luciferase-based assay, only seven compounds confirmed their specific involvement in TEAD activity. IA5 bearing a p-quinoid structure reduced the cytoplasmic level of phosphorylated YAP and the YAP–TEAD complex transcriptional activity and reduced cancer cell growth. IA5 is a promising hit compound for TEAD activity modulator development.

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.

The Hippo pathway is an important organ size control signaling network and the major regulatory mechanism of cell-contact inhibition. Yes associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are its targets and terminal effectors: inhibition of the pathway promotes YAP/TAZ translocation to the nucleus, where they interact with transcriptional enhancer associate domain (TEAD) transcription factors and coactivate the expression of target genes, promoting cell proliferation. Defects in the pathway can result in overgrowth phenotypes due to deregulation of stem-cell proliferation and apoptosis; members of the pathway are directly involved in cancer development. The pharmacological regulation of the pathway might be useful in cancer prevention, treatment, and regenerative medicine applications; currently, a few compounds can selectively modulate the pathway. In this review, we present an overview of the Hippo pathway, the sequence and structural analysis of YAP/TAZ, the known ...

Allosteric peptide inhibitors of thymidylate synthase (hTS) bind to the dimer interface and stabilize the inactive form of the protein. Four interface residues were mutated to alanine, and interaction studies were employed to decode the key role of these residues in the peptide molecular recognition. This led to the identification of three crucial interface residues F59, L198, and Y202 that impart activity to the peptide inhibitors and suggest the binding area for further inhibitor design.

Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.

Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

We report on the development of a multilayer coated reflection-type long period fiber grating (LPG) biosensor, useful for the detection of antibiotic resistance bacteria. A standard LPG is first transformed in a more practical probe working in reflection mode, then it is coated by a primary layer of aPS and a secondary layer of PMMA in order to increase its surrounding refractive index sensitivity and at the same time provide the necessary conditions for a correct bio-functionalization. Standard linkage chemistry has been applied to anchor the bioreceptors on the probe surface. We show some preliminary results demonstrating the capability of our LPG biosensor to successfully monitor all the biological steps of the biomolecular experiments, including β-lactamase binding detection tests.

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.