Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Pre-clinical and clinical development of leukemia stem cell inhibitors in acute leukemias

In Leukemias, recent developments have demonstrated that the Hedgehog pathway plays a key-role in the peculiar ability of self renewal of leukemia stem cells. The aim of this research activity was to investigate, through a first in man, Phase I, open label, clinical trial, the role and the impact, mainly in terms of safety profile, adverse events and pharmacokinetics, of a Sonic Hedgehog inhibitor compound on a population of heavely pretreated patients affected by AML, CML, MF, or MDS, resistant or refractory to standard chemotherapy. Thirty-five patients have been enrolled. The drug was administered orally, in 28 days cycles, without rest periods. The compound showed a good safety profile. The half life was of 17-35 hours, justifying the daily administration. Significant signs of activity, in terms of reduction of bone marrow blast cell amount were seen in most of the patients enrolled. Interestingly, correlative biological studies demonstrated that, comparing the gene expression profyiling signature of separated CD34+ cells before and after one cycle of treatment, the most variably expressed genes were involved in the Hh pathway. Moreover, we observed that many genes involved in MDR (multidrug resistance)were significantly down regulated after treatment. These data might lead to future clinical trials based on combinatory approaches, including, for instance, Hh inhibitors and conventional chemotherapy.

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

Histone deacetylase inhibitors in adult patients with diffuse large b-cell lymphoma relapsed/refractory: a phase II study with Panobinostat

Backgrounds:Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Panobinostat showed encouraging therapeutic activity in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies.Purpose:FIL-PanAL10 (NCT01523834) is a phase II, prospective multicenter trial of the Fondazione Italiana Linfomi (FIL) to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL patients and to evaluate a possible relationships between response and any biological features. Patients and Methods:Patients with R/R DLBCL were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR); secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient’s specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. ResultsThirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively. Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR

Interactions of G-quadruplex binders and Topoisomerase I inhibitors in cancer cells

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

Natural compounds Camptothecin and Triptolide: highly specific enzyme inhibitors and tools to dissect transcriptional functions

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.

Assessment of molecular sequences and enzyme activity of amylase trypsin inhibitors (ATIs) from a selection of ancient and modern grains

Wheat amylase-trypsin inhibitors (ATIs) are a family of wheat proteins, which play an important role in plant defence against pest attacks. ATIs are also of great interest for their impact on human health and recently ATIs have been identified as major stimulators of innate immune cells. In this study, ten selected wheat samples with different ploidy level and year of release were used for the agronomic trial, for in vitro enzymatic assays and for ATIs gene sequencing. Wheat samples were grown under organic farming management during three consecutive cropping years at two growing areas (Italy and USA). The PCA analysis performed on the deduced amino acid sequences of four representative ATIs genes (WMAI, WDAI, WTAI-CM3, CMx) evidenced that the ten wheat varieties can be differentiated on the basis of their ploidy level, but not with respect to ancient or recently developed wheat genotypes. The results from in vitro alpha-amylase and trypsin inhibitory activities showed high variability among the ten wheat genotypes and the contribution of the genotype and the cropping year was significant for both inhibitory activities. The hexaploid wheat genotypes showed the highest inhibitory activities. Einkorn showed a very low or even absent alpha-amylase inhibitory activity and the highest trypsin inhibitory activity. It was not possible to differentiate ancient and recently developed wheat genotypes on the basis of their ATIs activity. The weather conditions differently affected the two inhibitory activities. In both cultivation areas, higher precipitation and lower high mean temperatures correlated with lower alpha-amylase inhibitory activities, while there were different correlations considering trypsin inhibitory activity for the two growing areas. The protein content negatively correlated with both inhibitory activities in USA and Italy. This information can be important in the understanding of plant defence mechanisms in relation to the effect of both genotype and abiotic and biotic stress.

Design and Synthesis of Novel Kinase Inhibitors for the Treatment of Chronic Respiratory Diseases

In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this innovative approach in the design of new ROCKs inhibitors.

TAZ role in lung cancer: resistance to BET inhibitors and functional interaction with lncRNA

Despite extensive research and introduction of innovative therapy, lung cancer prognosis remains poor, with a five years survival of only 17%. The success of pharmacological treatment is often impaired by drug resistance. Thus, the characterization of response mechanisms to anti-cancer compounds and of the molecular mechanisms supporting lung cancer aggressiveness are crucial for patient’s management. In the first part of this thesis, we characterized the molecular mechanism behind resistance of lung cancer cells to the Inhibitors of the Bromodomain and Extraterminal domain containing Proteins (BETi). Through a CRISPR/Cas9 screening we identified three Hippo Pathway members, LATS2, TAOK1 and NF2 as genes implicated in susceptibility to BETi. These genes confer sensitivity to BETi inhibiting TAZ activity. Conversely, TAZ overexpression increases resistance to BETi. We also displayed that BETi downregulate both YAP, TAZ and TEADs expression in several cancer cell lines, implying a novel BETi-dependent cytotoxic mechanism. In the second part of this work, we attempted to characterize the crosstalk between the TAZ gene and its cognate antisense long-non coding RNA (lncRNA) TAZ-AS202 in lung tumorigenesis. As for TAZ downregulation, TAZ-AS202 silencing impairs NSCLC cells proliferation, migration and invasion, suggesting a pro-tumorigenic function for this lncRNA during lung tumorigenesis. TAZ-AS202 regulates TAZ target genes without altering TAZ expression or localization. This finding implies an uncovered functional cooperation between TAZ and TAZ-AS202. Moreover, we found that the EPH-ephrin signaling receptor EPHB2 is a downstream effector affected by both TAZ and TAZ-AS202 silencing. EPHB2 downregulation significantly attenuates cells proliferation, migration and invasion, suggesting that, at least in part, TAZ-AS202 and TAZ pro-oncogenic activity depends on EPH-ephrin signaling final deregulation. Finally, we started to dissect the mechanism underlying the TAZ-AS202 regulatory activity on EPHB2 in lung cancer, which may involve the existence of an intermediate transcription factor and is the object of our ongoing research.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

Design, synthesis and biological evaluation of dual inhibitors of GSK-3B and Fyn kinases for the study of inflammatory pathways in neurodegenerative diseases

Neuroinflammation represents a key hallmark of neurodegenerative diseases and is the result of a complex network of signaling cascades within microglial cells. A positive feedback loop exists between inflammation, microglia activation and protein misfolding processes, that, together with oxidative stress and excitotoxicity, lead to neuronal degeneration. Therefore, targeting this vicious cycle can be beneficial for mitigating neurodegeneration and cognitive decline in central nervous system disorders. At molecular level, GSK-3B and Fyn kinases play a crucial role in microglia activation and their deregulation has been associated to many neurodegenerative diseases. Thus, we envisioned their combined targeting as an effective approach to disrupt this toxic loop. Specifically in this project, a hit compound, based on a 7-azaindole-3-aminothiazole structure, was first identified in a virtual screening campaign, and displayed a weak dual inhibitory activity on GSK-3B and Fyn, unbalanced towards the former. Then, in a commitment to uncover the structural features required for modulating the activity on the two targets, we systematically manipulated this compound by inserting various substitution patterns in different positions. The most potent compounds obtained were advanced to deeper investigations to test their ability of tackling the inflammatory burden also in cellular systems and to unveil their binding modes within the catalytic pocket. The new class of molecules synthesized emerged as a valuable tool to deepen our understanding of the complex network governing the inflammatory events in neurodegenerative disorders.