A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIß and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.

Identification of a new family of pyrazolo[3,4-d]pyrimidine derivatives as multitarget Fyn-Blk-Lyn inhibitors active on B- and T-lymphoma cell lines.

Abnormal activation of B-cell receptor (BCR) signaling plays a key role in the development of lymphoid malignancies, and could be reverted by the simultaneous inhibition of Lyn, Fyn and Blk, three members of the Src family kinase (SFK). Fyn and Blk are also promising targets for the treatment of some forms of T-cell non-Hodgkin lymphoma which point to the druggability of SFKs for the treatment of these cancers. We recently identified Si308 as a potent Fyn inhibitor, while preliminary data showed that it might also inhibit Lyn and Blk. Here, molecular modelling studies were coupled with enzymatic assays to further investigate the effect of Si308 on Lyn and Blk. A small library of pyrazolo[3,4-d]pyrimidines structurally related to Si308 was synthesized and tested on human lymphoma cell lines. Compound 2h emerged as a new multitarget inhibitor of Lyn, Fyn and Blk endowed with remarkable antiproliferative effects on human B and T lymphoma cell lines. Its favorable ADME properties make the compound suitable for further developments.

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.

Identification of new pyrrolo[2,3-d]pyrimidines as Src tyrosine kinase inhibitors in vitro active against Glioblastoma.

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC50 value of 7.1 µM.

Discovery of Multitarget Agents Active as Broad-Spectrum Antivirals and Correctors of Cystic Fibrosis Transmembrane Conductance Regulator for Associated Pulmonary Diseases.

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.

Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.

DNA Polymerases λ and ß: The Double-Edged Swords of DNA Repair.

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases ß and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors.

Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line.

Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity.

A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant.

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

Exploring the chemical space around the privileged pyrazolo[3,4-d]pyrimidine scaffold: toward novel allosteric inhibitors of T315I-mutated Abl.

A library of pyrazolo[3,4-d]pyrimidines, endowed with a high level of molecular diversity, has been developed applying a synthetic sequence that allowed C3, N1, C4, and C6 substitution. The enzymatic screening of this "privileged scaffold"-based compound collection, validated the use of a diversity-oriented approach in a field characteristically explored by target-oriented synthesis. In fact, several compounds showed high activity against the selected kinases (i.e., Src, Abl wt, and T315I mutated-form), furthermore and interestingly a new compound has emerged as an allosteric inhibitor of the T315I mutated-form of Abl, opening up new opportunities for the development of a novel class of noncompetitive inhibitors of Abl (T315I).

Identification of Hck inhibitors as hits for the development of antileukemia and anti-HIV agents.

Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein tyrosine kinases. High levels of Hck are associated with drug resistance in chronic myeloid leukemia. Furthermore, Hck activity has been connected with HIV-1. Herein, structure-based drug design efforts were aimed at identifying novel Hck inhibitors. First, an in-house library of pyrazolo[3,4-d]pyrimidine derivatives, which were previously shown to be dual Abl and c-Src inhibitors, was analyzed by docking studies within the ATP binding site of Hck to select the best candidates to be tested in a cell-free assay. Next, the same computational protocol was applied to screen a database of commercially available compounds. As a result, most of the selected compounds were found active against Hck, with Ki values ranging from 0.14 to 18.4 µM, confirming the suitability of the computational approach adopted. Furthermore, selected compounds showed an interesting antiproliferative activity profile against the human leukemia cell line KU-812, and one compound was found to block HIV-1 replication at sub-toxic concentrations.

A combination strategy to inhibit Pim-1: synergism between noncompetitive and ATP-competitive inhibitors.

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.

Pyrazolo[3,4-d]pyrimidine Prodrugs: Strategic Optimization of the Aqueous Solubility of Dual Src/Abl Inhibitors.

Design and synthesis of prodrugs of promising drug candidates represents a valid strategy to overcome the lack of favorable ADME properties, in particular aqueous solubility and bioavailability. We report herein the successful application of this strategy with two representative pyrazolo[3,4-d]pyrimidine derivatives (1 and 2), which led to the development of the corresponding and highly water-soluble antitumor prodrugs (7 and 8). In vitro studies confirmed a significant improvement of aqueous solubility and, for compound 8, good plasma stability, suggesting superior in vivo bioavailability. As expected, the uncleaved water-soluble prodrugs 7 and 8 showed no activity toward the enzymatic targets (c-Src and c-Abl) but revealed promising antiproliferative activity in myeloid cell lines, as a consequence of the in vitro hydrolysis of the selected solubilizing moiety, followed by the release of the active compounds (1 and 2).