Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against Both Diseases

GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg-) Salmonella Typhimurium GMMA. Only the OAg- GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg- GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg- GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors

Expanding the Arsenal of FGFR Inhibitors: A Novel Chloroacetamide Derivative as a New Irreversible Agent With Anti-proliferative Activity Against FGFR1-Amplified Lung Cancer Cell Lines

Fibroblast Growth Factor Receptors (FGFR1–4) have a critical role in the progression of several human cancers, including Squamous Non-Small-Cell Lung Cancer (SQCLC). Both non-selective and selective reversible FGFR inhibitors are under clinical investigation for the treatment of patients with tumors harboring FGFR alterations. Despite their potential efficacy, the clinical development of these drugs has encountered several challenges, including toxicity, and the appearance of drug resistance. Recent efforts have been directed at development of irreversible FGFR inhibitors, which have the potential to exert superior anti-proliferative activity in tumors carrying FGFR alterations. With this in mind, we synthetized, and investigated a set of novel inhibitors possessing a warhead potentially able to covalently bind a cysteine in the P-loop of FGFR. Among them, the chloroacetamide UPR1376 resulted able to irreversible inhibit FGFR1 phosphorylation in FGFR1 over-expressing cells generated from SQCLC SKMES-1 cells. In addition, this compound inhibited cell proliferation in FGFR1-amplified H1581 cells with a potency higher than the reversible inhibitor BGJ398 (infigratinib), while sparing FGFR1 low-expressing cells. The anti-proliferative effects of UPR1376 were demonstrated in both 2D and 3D systems and were associated with the inhibition of MAPK and AKT/mTOR signaling pathways. UPR1376 inhibited cell proliferation also in two BGJ398-resistant cell clones generated from H1581 by chronic exposure to BGJ398, although at concentrations higher than those effective in the parental cells, likely due to the persistent activation of the MAPK pathway associated to NRAS amplification. Combined blockade of FGFR1 and MAPK signaling, by UPR1376 and trametinib respectively, significantly enhanced the efficacy of UPR1376, providing a means of circumventing resistance to FGFR1 inhibition. Our findings suggest that the insertion of a chloroacetamide warhead on a suitable scaffold, as exemplified by UPR1376, is a valuable strategy to develop a novel generation of FGFR inhibitors for the treatment of SQCLC patients with FGFR alterations

Un approccio integrato per la caratterizzazione delle proprietà chimico-fisiche di NCEs in fase di discovery

Negli ultimi vent’anni le piccole molecole sviluppate come nuovi farmaci hanno mostrato una complessità chimica crescente in confronto a quelle ottimizzate prima del 2000. Un esempio di questa tendenza è offerto dagli anfoliti, la cui caratterizzazione chimica e biologica è complessa. In tale contesto, lo scopo di questa tesi di dottorato è la definizione di un flusso di lavoro per la caratterizzazione delle proprietà chimico-fisiche di nuovi candidati molecolari allo stadio di ricerca o di primo sviluppo. L’approccio sperimentale include l’analisi di costanti di ionizzazione (pKa) lipofilia (log P or log D7.4) e solubilità ed è finalizzato alla caratterizzazione di composti disponibili allo stato solido (i.e. polveri). Tuttavia, sono inclusi saggi rapidi per l’analisi preliminare di candidati molecolari esclusivamente accessibili come soluzioni in dimetilsolfossido. Prima di essere applicate a nuove entità chimiche, le tecniche sperimentali sono state valutate su composti commerciali, così da definirne applicabilità, vantaggi e limiti. L’ approccio definito per la caratterizzazione chimico-fisica si è dimostrato utile alle esigenze pratiche di un dipartimento di ricerca pre-clinica, restituendo dati funzionali alle prime fasi di sviluppo (e.g. indicazioni di massima per le condizioni di salificazione e di ottimizzazione della forma solida del principio attivo). In aggiunta i risultati sperimentali di questo lavoro saranno inclusi nel set di calibrazione di software ad uso interno per la predizione di proprietà molecolari (pKas, lipofilia) e per la correlazione di dati in-vitro-in-vivo (solubilità). Di fatto tale strategia è la chiave per sviluppare sistemi di calcolo esperti, ragione per cui la determinazione sperimentale di proprietà chimico-fisiche è un’esigenza ancora attuale nella ricerca farmaceutica.In the last 20 years, small molecules developed as potential new drugs have shown an increasing chemical complexity in comparison to compounds optimized before the year 2000. Current research trend is well exemplified by ampholytes, for which molecular and biological characterisation is anything but trivial. In this context, the aim of the present Ph.D. thesis was to outline an in-house integrated work-flow for physico-chemical profiling of new drug candidates at discovery and early development stage. Screening included ionization constants (pKas), lipophilicity (log P or log D7.4) and solubility. This approach was expected to be applied to new chemical entities freely accessible in solid form as well as to include medium/high-throughput preliminary assays suitable for those candidates that were exclusively available as DMSO stock solutions. Before transferring the techniques to actual drug candidates, those were tested on commercially available standards to evaluate their feasibility, advantages and drawbacks. This in-house screening of physico-chemical properties provided reliable data for pre-development (e.g. guidelines for salt screening and solid form optimization). Moreover, experimental results from the present work will be included in the calibrating set of programs for in-house prediction of molecular properties (pKas, lipophilicity) and for in-vitro-in-vivo correlation (solubility). In fact, inclusion of discovery compounds is the key to develop expert systems and that is why experimental assays for measuring physico-chemical parameters are still warranted

Combined targeting of fatty acid amide hydrolase and melatonin receptors promotes neuroprotection and stimulates inflammation resolution in rats

Background and purpose: Devising novel strategies to therapeutically favour inflammation resolution and provide neuroprotection is an unmet clinical need. Enhancing endocannabinoid tone by inhibiting the catabolic enzyme fatty acid amide hydrolase (FAAH), or stimulating melatonin receptors has therapeutic potential to treat neuropathological states in which neuroinflammation plays a central role. Experimental approach: A rodent hippocampal explant model of inflammatory injury was used to assess the effects of UCM1341, a dual-acting compound with FAAH inhibitory action and agonist activity at melatonin receptors, against neuroinflammatory damage. FAAH activity was measured by a radiometric assay, and N-acylethanolamine levels were assessed by HPLC-MS/MS methods. FAAH distribution, evolution of inflammation and the contribution of UCM1341 to the expression of proteins controlling macrophage behaviour were investigated by biochemical and confocal analyses. Key results: UCM1341 exhibited greater neuroprotection against neuroinflammatory degeneration, compared with the reference compounds URB597 (FAAH inhibitor) and melatonin. During neuroinflammation, UCM1341 augmented the levels of anandamide and N-oleoylethanolamine, but not N-palmitoylethanolamine, up-regulated PPAR-α levels, attenuated demyelination and prevented the release of TNF-α. UCM1341 modulated inflammatory responses by contributing to microglia/macrophage polarization, stimulating formation of lipid-laden macrophages and regulating expression of proteins controlling cholesterol metabolism and efflux. The neuroprotective effects of UCM1341 were prevented by PPARα, TRPV1 and melatonin receptor antagonists. Conclusion and implications: UCM1341, by enhancing endocannabinoid and melatoninergic signalling, benefits neuroprotection and stimulates inflammation resolution pathways. Our findings provide an encouraging prospect of therapeutically targeting endocannabinoid and melatoninergic systems in inflammatory demyelinating states in the CNS

Protein-Protein Interaction Inhibitors Targeting the Eph-Ephrin System with a Focus on Amino Acid Conjugates of Bile Acids

The role of the Eph-ephrin system in the etiology of pathological conditions has been consolidated throughout the years. In this context, approaches directed against this signaling system, intended to modulate its activity, can be strategic therapeutic opportunities. Currently, the most promising class of compounds able to interfere with the Eph receptor-ephrin protein interaction is composed of synthetic derivatives of bile acids. In the present review, we summarize the progresses achieved, in terms of chemical expansions and structure-activity relationships, both in the steroidal core and the terminal carboxylic acid group, along with the pharmacological characterization for the most promising Eph-ephrin antagonists in in vivo settings

Identification of Human Alanine-Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5'-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity

Cell-targeted c(AmpRGD)-sunitinib molecular conjugates impair tumor growth of melanoma

Drug resistance and off-organ toxicity remain unsolved issues in chemotherapy of advanced-stage melanoma patients. Thus, the creation of new molecular conjugates able to combine a selective accumulation, high ability of internalization and signaling pathway inhibition, are highly requested. Recently, we reported a new class of molecular conjugates, compounds 1–3, where the anti-αVβ3 integrin peptidomimetic c(AmpRGD), which is a selective ligand for αVβ3 integrin, was covalently bound to the tyrosine kinase inhibitor sunitinib. Here, we report that these c(AmpRGD)-sunitinib conjugates and, in particular, compound 3, are selectively internalized by human melanoma cells through αVβ3 receptor-mediated endocytosis. Compound 3 is more effective than sunitinib in reducing in vitro melanoma cells proliferation, cloning efficiency, migration, and invasion. More interestingly, compound 3 is able to significantly reduce the growth of xenografted melanoma tumor developed in immune-compromised mice, more efficiently than an equimolar dose of sunitinib. Indeed, its targeting ability was demonstrated by the selective localization at the tumor level with respect to healthy tissues. Thus, c(AmpRGD)-sunitinib conjugates such as compound 3 could serve as intriguing multiple-target agents to selectively reach melanoma cells and interfere with the progression of the disease