Structural and biophysical characterization of RAD52 as novel pharmacological target for synthetic lethality

RAD52 is a protein involved in various DNA reparation mechanisms. In the last few years, RAD52 has been proposed as a novel pharmacological target for cancer synthetic lethality strategies. Hence, this work has the purpose to investigate RAD52 protein, with biophysical and structural tools to shed light on proteins features and mechanistic details that are, up to now poorly described, and to design novel strategies for its inhibition. My PhD work had two goals: the structural and functional characterization of RAD52 and the identification of novel RAD52 inhibitors. For the first part, RAD52 was characterized both for its DNA interaction and oligomerization state together with its propensity to form high molecular weight superstructures. Moreover, using EM and Cryo-EM techniques, additional RAD52 structural hallmarks were obtained, valuable both for understanding protein mechanism of action and for drug discovery purpose. The second part of my PhD project focused on the design and characterization of novel RAD52 inhibitors to be potentially used in combination therapies with PARPi to achieve cancer cells synthetic lethality, avoiding resistance occurrence and side effects. With this aim we selected and characterized promising RAD52 inhibitors through three different approaches: 19F NMR fragment-based screening; virtual screening campaign; aptamers computational design. Selected hits (fragments, molecules and aptamers) were investigated for their binding to RAD52 and for their mechanism of inhibition. Collected data highlighted the identification of hits worthy to be developed into more potent and selective RAD52 inhibitors. Finally, a side project carried out during my PhD is reported. GSK-3β protein, an already validated pharmacological target was investigated using biophysical and structural biology tools. Here, an innovative and adaptable drug discovery screening pipeline able to directly identify selective compounds with binding affinities not higher than a reference binder was developed

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives

Background: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. Methods: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). Results: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. Conclusions: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach

Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer’s disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition

GSK-3β Allosteric Inhibition: A Dead End or a New Pharmacological Frontier?

Most kinase inhibitors are designed to bind to highly homologous ATP-binding sites, which leads to promiscuity and possible off-target effects. Allostery is an alternative approach to pursuing selectivity. However, allostery is difficult to exploit due to the wide variety of underlying mechanisms and the potential involvement of long-range conformational effects that are difficult to pinpoint. GSK-3β is involved in several pathologies. This critical target has an ATP-binding site that is highly homologous with the orthosteric sites of other kinases. Unsurprisingly, there is also great similarity between the ATP-binding sites of GSK-3β and its isomer, which is not redundant and thus would benefit from selective inhibition. Allostery would also allow for a moderate and tunable inhibition, which is ideal for GSK-3β, because this target is involved in multiple pathways, some of which must be preserved. However, despite considerable research efforts, only one allosteric GSK-3β inhibitor has reached the clinic. Moreover, unlike other kinases, there are no X-ray structures of GSK-3β in complex with allosteric inhibitors in the PDB data bank. This review aims to summarize the state of the art in allosteric GSK-3β inhibitor investigations, highlighting the aspects that make this target challenging for an allosteric approach

GSK3β as a novel promising target to overcome chemoresistance in pancreatic cancer

Pancreatic cancer is an aggressive malignancy with increasing incidence and poor prognosis due to its late diagnosis and intrinsic chemoresistance. Most pancreatic cancer patients present with locally advanced or metastatic disease characterized by inherent resistance to chemotherapy. These features pose a series of therapeutic challenges and new targets are urgently needed. Glycogen synthase kinase 3 beta (GSK3β) is a conserved serine/threonine kinase, which regulates key cellular processes including cell proliferation, DNA repair, cell cycle progression, signaling and metabolic pathways. GSK3β is implicated in non-malignant and malignant diseases including inflammation, neurodegenerative diseases, diabetes and cancer. GSK3β recently emerged among the key factors involved in the onset and progression of pancreatic cancer, as well as in the acquisition of chemoresistance. Intensive research has been conducted on key oncogenic functions of GSK3β and its potential as a druggable target; currently developed GSK3β inhibitors display promising results in preclinical models of distinct tumor types, including pancreatic cancer. Here, we review the latest findings about GSK-3β biology and its role in the development and progression of pancreatic cancer. Moreover, we discuss therapeutic agents targeting GSK3β that could be administered as monotherapy or in combination with other drugs to surmount chemoresistance. Several studies are also defining potential gene signatures to identify patients who might benefit from GSK3β-based therapeutic intervention. This detailed overview emphasizes the urgent need of additional molecular studies on the impact of GSK3β inhibition as well as structural analysis of novel compounds and omics studies of predictive biomarkers

Identification of Serogroups Australis and Icterohaemorrhagiae in Two Dogs with a Severe Form of Acute Leptospirosis in Italy

Leptospirosis is an infectious disease that causes serious illness in dogs. For this reason, epidemiological and clinical studies focusing on disease characterization are widely advocated. The aim of this study was to characterize the leptospires identified in dogs with confirmed symptomatic acute leptospirosis. Leptospira spp. DNA detected in urine, blood, or both samples from nine infected dogs was analyzed using the multi-locus sequence typing (MLST) technique. Leptospires from two dogs were successfully typed: one was identified as belonging to Sequence Type (ST) 17 and one to ST198, both within the L. interrogans species, serogroups Icterohaemorrhagiae and Australis, respectively. Based on the results of routine serologic tests, antibodies reactive toward these serogroups are commonly revealed in dogs in Italy. This study provides the first molecular analysis that identifies infecting Leptospira directly on DNA from biological samples of dogs, showing that serogroup Australis can lead to a severe clinical presentation of leptospirosis in infected dogs

Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer’s disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition

RX-3117 (fluorocyclopentenyl cytosine): a novel specific antimetabolite for selective cancer treatment

Introduction: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types

ARN25068, a versatile starting point towards triple GSK-3β/FYN/DYRK1A inhibitors to tackle tau-related neurological disorders

The human kinome plays a crucial role in several pathways. Its dysregulation has been linked to diverse central nervous system (CNS)-related disorders with a drastic impact on the aging population. Among them, tauopathies, such as Alzheimer's Disease (AD) and Frontotemporal Lobar degeneration (FTLD-tau), are neurodegenerative disorders pathologically defined by the presence of hyperphosphorylated tau-positive intracellular inclusions known as neurofibrillary tangles (NFTs). Compelling evidence has reported the great potential of the simultaneous modulation of multiple protein kinases (PKs) involved in abnormal tau phosphorylation through a concerted pharmacological approach to achieve a superior therapeutic effect relative to classic "one target, one drug" approaches. Here, we report on the identification and characterization of ARN25068 (4), a low nanomolar and well-balanced dual GSK-3β and FYN inhibitor, which also shows inhibitory activity against DYRK1A, an emerging target in AD and tauopathies. Computational and X-Ray studies highlight compound 4's typical H-bonding pattern of ATP-competitive inhibitors at the binding sites of all three PKs. In a tau phosphorylation assay on Tau0N4R-TM-tGFP U2OS cell line, 4 reduces the extent of tau phosphorylation, promoting tau-stabilized microtubule bundles. In conclusion, this compound emerges as a promising prototype for further SAR explorations to develop potent and well-balanced triple GSK-3β/FYN/DYRK1A inhibitors to tackle tau hyperphosphorylation