Gianpiero Garau

Thiazides are among the most efficacious and commonly used drugs for the treatment of hypertension. The nanomolar stabilizer N3SA binds specifically to the recently discovered thiazide-binding site of the membrane target NAPE-PLD, showing sustained arterial blood pressure-lowering effects and vasodilation in spontaneous hypertensive rats (SHRs). To further support the relation between stabilizers anchored to NAPE-PLD and their beneficial effects on hypertension, we selected compound analogues of N3SA with chemical modifications at the three target-interacting sulfonic groups, including the drug Suramin. Each compound was injected i.v in an adult SHR (systolic blood pressure of 217 ± 5 mmHg) to evaluate the frequency components contribution to cerebral and peripheral arteriolar vasomotion. We visualized the pial and rectus femoral muscle microcirculation by Epi-illumination, measuring changes in the rhythmic arteriolar diameter. Findings showed that the minor structural differences in compounds correlated with the contribution of the six different frequency components affecting the arterial tone, as well as their vasodilatory effects, in both cerebral and femoral muscle arterioles. These results provide evidence that the spectra analysis of the regulation mechanisms of vascular tone and arterial blood pressure can accurately reflect the structure–activity correlations of different analogues of an antihypertensive compound.

The present State of the Art Review will take stock of targeting the endocannabinoid system (ECS) in the management of hypertension and vascular diseases. Major efforts have been made in the last thirty years to develop compounds modulating the ECS for diseases, both in the central and peripheral tissues. Agonists of the cannabinoid receptor CB1 elicited hypotension but were at strong risks of inducing tachycardia, heart and kidney damage. The clinical translation of CB2 agonists, as well as antiobesity CB1 blockers, proposed as promising treatments for cardiovascular risk factors, was complicated by adverse side effects. Inhibitors of the membrane enzyme fatty acid amide hydrolase that degrades the endocannabinoid anandamide normalized elevated blood pressure, at least in hypertensive rats. The turning point came when we discovered the phospholipase NAPE-PLD as a systemic target of thiazide diuretics, revealing physicians have indeed targeted the ECS for over sixty years in clinics, unknowingly. The membrane-associated target promotes diuretic effect through its internal channel and generates at the same time anandamide and other lipid signaling amides that exert marked protective actions, useful for the chronic treatment of hypertension. Results have extended our knowledge of the mechanism of thiazide medications, rationalizing both their acute and chronic therapeutic effects. The stabilization of NAPE-PLD by chemical agents that bind to the thiazide-binding site is efficacious in the treatment of hypertension and vascular resistance, providing a validated approach in clinics for the ECS modulation in human diseases associated with neurovascular alterations.

Background. It has been estimated that more than 50% of cancers are related to the dysregulation of the PI3K-PDK1-Akt signaling axis. Within this pathway, phosphoinositide-dependent kinase-1 (PDK1) and Aurora kinase A (AurA) have key roles. PDK1 is a phosphorylation-mediated activator of Akt, the main apoptotic regulator, thereby acting at the crossroads of several oncogenic pathways. Overexpression and/or aberrant activation of AurA have been functionally linked to neoplastic transformation principally through the development of centrosome amplification and chromosomal instability. In this study, the molecular mechanisms involved in the dual inhibitory capacity of PDK1- and AurA-targeting OXID-pyridonyl compounds were explored in a panel of tumor cell lines carrying PI3KCA, K-RAS, PTEN, RB-1, EGFR and/or TP53 mutations Materials and Methods. To evaluate the enzymatic inhibitory activity, the oxindole-based derivatives were subjected to FRET-based Z′-Lyte assay (Invitrogen™) against PDK1 and AurA. All in vitro assays for ADME-Tox profiling were performed as published previously[1]. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72-h exposures with increasing concentrations (from 0.014 to 30 µM) in a panel of 15 cell lines. Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK-1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds. Results. Among the four evaluated compounds, SA16 displayed nanomolar inhibitory potency against both PDK1 and AurA. In vitro, drugs SA16 and VI8 showed to exert anti-tumor activity in the 6647, TC71, SKNMC Ewing sarcoma (ES) cells lines and in the HCT116 colon carcinoma-derived cells (IC50s values between 2.6 and 25 µM after 72-h exposures), when tested in a panel of 15 cancer cell lines. These results were confirmed by the evaluation of 3D-clonogenic capacity in soft agar. The effect of 1 µM SA16 at the protein level was evaluated after 5 to 120 min exposure in the ES cell line SKNMC. No modulation of Aur-A protein or PDK-1 phosphorylation (Ser241) were observed in SKNMC cells, whereas a decrease of downstream PDK1 targets such as p-AKT, p-RSK2, and p-p44/42 MAPK was observed after 30 min of exposure. In addition, SA16 displayed acceptable in vitro ADME-Tox properties. Our results on structural insights into the mode of binding[2], in vitro drug-intake kinetics and in vivo PK appear to indicate that SA16 presents adequate features as a scaffold for the design of PDK1/AurA dual-target molecules. Conclusion. We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.[1] Runfola M. et al. Data Brief 29 (2020) 105206.[2] Sistito S. et al. Eu J Med Chem 226 (2021) 11389. Citation Format: Maria Eugenia Riveiro, Samuel Huguet, Ramiro Vazquez, Roberta Frapolli, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai, Gianpiero Garau, Simona Rapposelli. Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2569.

Because of the key relevance of protein–protein interactions (PPI) in diseases, the modulation of protein-protein complexes is of relevant clinical significance. The successful design of binding compounds modulating PPI requires a detailed knowledge of the involved protein-protein system at molecular level, and investigation of the structural motifs that drive the association of the proteins at the recognition interface. These elements represent hot spots of the protein binding free energy, define the complex lifetime and possible modulation strategies. Here, we review the advanced technologies used to map the PPI involved in human diseases, to investigate the structure-function features of protein complexes, and to discover effective ligands that modulate the PPI for therapeutic intervention.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane-penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody-based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo-EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to the static surface-exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody-based neutralization of PA and bring into focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is the main virulent factor of anthrax. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the toxin’s cytotoxic enzymes are extruded. The PA pore is formed by a cooperative conformational change where PA’s membrane-penetrating loops associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target of monoclonal antibodies-based therapy. cAb29 is a highly effective neutralizing antibody against PA. We determined the cryo-EM structure of PA in complex with the Fab portion of cAb29. We found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to a static region on PA’s surface, thereby preventing pore formation. Therefore, our results provide the structural basis for the antibody-based neutralization of PA and bring to focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug’s activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.