2,6-Diphenyl-imidazopyridine derivatives as novel prototypes of anticancer agents targeting aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH) superfamily comprises 19 different enzyme types located in specific subcellular districts, including cytosol and mitocondria. Their main function is to oxidize endogenous and exogenous aldehydes produced in human cells. In particular, isoforms 1A1, 1A2 and 1A3 catalyze the transformation of retinal into retinoic acid, which is a potent differentiation tissue factor for cellular development. Overexpression of these three isoforms in cancer stem cells (CSC), underlined in recent studies, is to date extremely important in cancer field, as it offers the chance to use these proteins both as prognostic marker and as novel targets in the fight against cancer. Here we present a novel series of 2,6-diphenyl-imidazol[1,2-a]pyridines, designed as aldehyde dehydrogenase inhibitors by means of a structured-based optimizations of a previously developed lead, GA11. The novel compounds were evaluated in vitro for their activity and selectivity against the three isoforms of the ALDH1A family, and investigated through crystallization and modeling studies for their ability to interact with the catalytic site of the 1A3 isoform. Tested in vitro on different populations of CSCs, obtained from glioma, colorectal and prostate tissue specimens, they exhibited a relevant anti-proliferative efficacy, thus paving the way for treating cancer by means of the still untapped aldehyde dehydrogenases

Design of small molecules interacting with wild-type and mutant rhodopsins

Rhodopsin is a class A member of the G protein-coupled receptor (GPCR) family that detects light in the rod photoreceptor cells, and initiates the visual transduction cascade through the isomerization of 11-cis to all-trans retinal. Rhodopsin mutations and altered concentration of the retinal cofactor are associated with retinitis pigmentosa (RP), a group of degenerative diseases that lead to progressive loss of photoreceptor cells and blindness. Mutations at the N-terminal tail of rhodopsin are the most common mutations associated with RP, causing receptor misfolding and mislocalization, while mutations at the C-terminal tail of rhodopsin cause mistrafficking from Golgi to the rod outer segment, and are notably associated with more aggressive RP forms, thus becoming of particular interests for the development of therapeutic strategies. Levels of endogenous retinal cofactor are finely controlled by a series of enzymes, including the aldehyde dehydrogenase (ALDH) 1A3 isoform. Although its role still unclear, ALDH1A3 enzymatic activity may correlate with retinal concentration, and its modulation may have therapeutic implications in RP 2. However, limited experimental evidence and the lack of structural details that may clarify the role of rhodopsin mutations and ALDH1A3 in RP currently hamper the drug design process. In this work, these limitations have been addressed using three different computational modelling approaches: The first approach is based on the evidence that the correct folding and localization of mutant rhodopsin can be partially rescued by small chaperone ligands, which are able to bind rhodopsin in the 11-cis retinal binding site. To identify new small molecules that could act as a molecular chaperone for misfolded P23H mutant rhodopsin, the homology model of P23H mutant rhodopsin was generated and used as rigid receptor for a docking-based virtual screening carried out on different libraries of compounds. Following the virtual screening procedure, candidate hits were selected based on a combination of docking score, docking pose, and polar/hydrophobic interactions with the receptor and they were further investigated for their ability to restore the correct localization of mutant rhodopsin on the cell membrane by experimental validation in cell-based assay. Interestingly, one molecule (i.e., compound A3) was found to restore the correct protein folding and membrane localization. The second approach describes a computational workflow for studying clinically relevant C-terminal rhodopsin mutants in RP and to provide structural insights into the molecular mechanisms involved in the trafficking of WT and P347 mutant rhodopsin to the rod membrane. The conformational space of full-length WT and P347 mutant rhodopsin was explored by molecular dynamics (MD) simulations. The WT VAPA-COOH motif adopts a unique conformation that is not found in pathological mutants, suggesting that structural features could better explain the pathogenicity of P347 rhodopsin mutants than physicochemical or steric determinants. Interestingly, MD simulations of isolated and N-capped deca-peptides corresponding to the C-terminal tail of WT and P347 mutant rhodopsin showed results that are highly comparable to the full-length system, suggesting that deca-peptides are reliable model systems for studying C-terminal rhodopsin mutations in silico. The last approach focuses on the role of ALDH1A3 in the metabolism of retinal cofactor. Here we hypothesize that inhibition of ALDH1A3 may increase the level of endogenous 11-cis retinal in RP, given that one of the causes of the disease is related to the low cellular levels of the retinal cofactor. Starting from the reference ALDH inhibitor GA11, previously developed by our group, a novel series of imidazo[1,2-a]pyridines was developed and optimized by means of a structure-based approach. These novel compounds were evaluated in vitro for their activity and selectivity against the ALDH1A family and investigated through X-ray crystallography and molecular modeling studies for their ability to interact with the catalytic site of the 1A3 isoform. Overall, computational approach provided structural and thermodynamics information that are essential for the design and optimization of small molecules ALDH1A3 inhibitors. As a side project of this thesis, the main sequence and structural features of the RNA-dependent RNA polymerase of emerging RNA viruses, including the most recent SARS-CoV-2, were reviewed and the main limitation that prevent the successful application of the structure-based drug design approach in antiviral drug discovery process were discussed 8. In this context, the repositioning of sofosbuvir, approved for HCV infections, as an anti-Flaviviruses drug, was proposed through experimental and computational evaluations. In general, the data collected in this thesis enhance the use of computational techniques in a context of multidisciplinary effort to improve drug design and drug discovery approach in different research fields

Conformational insights into the C-terminal mutations of human rhodopsin in retinitis pigmentosa

Rhodopsin is a light-sensitive transmembrane receptor involved in the visual transduction cascade. Among the several rhodopsin mutations related to retinitis pigmentosa (RP), those affecting the C-terminal VAPA-COOH motif that is implicated in rhodopsin trafficking from the Golgi to the rod outer segment are notably associated with more aggressive RP forms. However, molecular reasons for defective rhodopsin signaling due to VAPA-COOH mutations, which might include steric hindrance, physicochemical features and structural determinants, are yet unknown, thus limiting further drug design approaches. In this work, clinically relevant rhodopsin mutations at the P347 site within the VAPA-COOH motif were investigated by molecular dynamics (MD) simulations and compared to the wild-type (WT) system. In agreement with experimental evidence, conformational fluctuations of the intrinsically disordered C-terminal tail of WT and mutant rhodopsin were found not to affect the overall structure of the transmembrane domain, including binding to the retinal cofactor. The WT VAPA-COOH motif adopts a unique conformation that is not found in pathological mutants, suggesting that structural features could better explain the pathogenicity of P347 rhodopsin mutants than physicochemical or steric determinants. These results were confirmed by MD simulations in both membrane-embedded full-length opsin and membrane-free C-terminal deca-peptides, these latter becoming very useful and small-size model systems for further investigations of rhodopsin C-terminal mutations. Structural details elucidated in this work might facilitate the understanding of the pathological mechanisms of this class of rhodopsin mutants, which will be instrumental to the development of new therapeutic strategies

Targeting the RdRp of Emerging RNA Viruses: The Structure-Based Drug Design Challenge

The RNA-dependent RNA polymerase (RdRp) is an essential enzyme for the viral replication process, catalyzing the viral RNA synthesis using a metal ion-dependent mechanism. In recent years, RdRp has emerged as an optimal target for the development of antiviral drugs, as demonstrated by recent approvals of sofosbuvir and remdesivir against Hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. In this work, we overview the main sequence and structural features of the RdRp of emerging RNA viruses such as Coronaviruses, Flaviviruses, and HCV, as well as inhibition strategies implemented so far. While analyzing the structural information available on the RdRp of emerging RNA viruses, we provide examples of success stories such as for HCV and SARS-CoV-2. In contrast, Flaviviruses’ story has raised attention about how the lack of structural details on catalytically-competent or ligand-bound RdRp strongly hampers the application of structure-based drug design, either in repurposing and conventional approaches

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development

A selective competitive inhibitor of aldehyde dehydrogenase 1A3 hinders cancer cell growth, invasiveness and stemness in vitro

Aldehyde dehydrogenase 1A3 (ALDH1A3) belongs to an enzymatic superfamily composed by 19 different isoforms, with a scavenger role, involved in the oxidation of a plethora of aldehydes to the respective carboxylic acids, through a NAD+‐dependent reaction. Previous clinical studies highlighted the high expression of ALDH1A3 in cancer stem cells (CSCs) correlated to a higher risk of cancer relapses, chemoresistance and a poor clinical outcome. We report on the structural, biochemical, and cellular characterization of NR6, a new selective ALDH1A3 inhibitor derived from an already published ALDH non‐selective inhibitor with cytotoxic activity on glioblastoma and colorectal cancer cells. Crystal structure, through X‐Ray analysis, showed that NR6 binds a non‐conserved tyrosine residue of ALDH1A3 which drives the selectivity towards this isoform, as supported by computational binding simulations. Moreover, NR6 shows anti‐metastatic activity in wound healing and invasion assays and induces the downregulation of cancer stem cell markers. Overall, our work confirms the role of ALDH1A3 as an important target in glioblastoma and colorectal cells and propose NR6 as a promising molecule for future preclinical studies

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition