A benchmarking of pipelines for detecting ncRNAs from RNA-Seq data.

Next-Generation Sequencing (NGS) is a high-throughput technology widely applied to genome sequencing and transcriptome profiling. RNA-Seq uses NGS to reveal RNA identities and quantities in a given sample. However, it produces a huge amount of raw data that need to be preprocessed with fast and effective computational methods. RNA-Seq can look at different populations of RNAs, including ncRNAs. Indeed, in the last few years, several ncRNAs pipelines have been developed for ncRNAs analysis from RNA-Seq experiments. In this paper, we analyze eight recent pipelines (iSmaRT, iSRAP, miARma-Seq, Oasis 2, SPORTS1.0, sRNAnalyzer, sRNApipe, sRNA workbench) which allows the analysis not only of single specific classes of ncRNAs but also of more than one ncRNA classes. Our systematic performance evaluation aims at guiding users to select the appropriate pipeline for processing each ncRNA class, focusing on three key points: (i) accuracy in ncRNAs identification, (ii) accuracy in read count estimation and (iii) deployment and ease of use.

Stereoselective synthesis of 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-one derivatives as PIM kinase inhibitors inspired from marine alkaloids.

We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.

Identification of unprecedented ATP-competitive choline kinase inhibitors.

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

Comprehensive kinome NGS targeted expression profiling by KING-REX.

BACKGROUND: Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human 'kinome', demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets. RESULTS: In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3'- and 5'-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA. CONCLUSIONS: These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases.

Phase I dose escalation study of NMS-1286937, an orally available Polo-Like Kinase 1 inhibitor, in patients with advanced or metastatic solid tumors.

Background Pharmacological inhibition of polo-like kinase 1 (PLK1) represents a new approach for the treatment of solid tumors. This study was aimed at determining the first cycle dose-limiting toxicities (DLTs) and related maximum tolerated dose (MTD) of NMS-1286937, a selective ATP-competitive PLK1-specific inhibitor. Secondary objectives included evaluation of its safety and pharmacokinetic (PK) profile in plasma, its antitumor activity, and its ability to modulate intracellular targets in biopsied tissue. Methods This was a Phase I, open-label, dose-escalation trial in patients with advanced/metastatic solid tumors. A treatment cycle comprised 5 days of oral administration followed by 16 days of rest, for a total of 21 days (3-week cycle). Results Nineteen of 21 enrolled patients with confirmed metastatic disease received study medication. No DLTs occurred at the first 3 dose levels (6, 12, and 24 mg/m2/day). At the subsequent dose level (48 mg/m2/day), 2 of 3 patients developed DLTs. An intermediate level of 36 mg/m2/day was therefore investigated. Four patients were treated and two DLTs were observed. After further cohort expansion, the MTD and recommended phase II dose (RP2D) were determined to be 24 mg/m2/day. Disease stabilization, observed in several patients, was the best treatment response observed. Hematological toxicity (mostly thrombocytopenia and neutropenia) was the major DLT. Systemic exposure to NMS-1286937 increased with dose and was comparable between two cycles of treatment following oral administration of the drug. Conclusions This study successfully identified the MTD and DLTs for NMS-1286937 and characterized its safety profile.

KAOS: a new automated computational method for the identification of overexpressed genes.

BACKGROUND: Kinase over-expression and activation as a consequence of gene amplification or gene fusion events is a well-known mechanism of tumorigenesis. The search for novel rearrangements of kinases or other druggable genes may contribute to understanding the biology of cancerogenesis, as well as lead to the identification of new candidate targets for drug discovery. However this requires the ability to query large datasets to identify rare events occurring in very small fractions (1-3 %) of different tumor subtypes. This task is different from what is normally done by conventional tools that are able to find genes differentially expressed between two experimental conditions. RESULTS: We propose a computational method aimed at the automatic identification of genes which are selectively over-expressed in a very small fraction of samples within a specific tissue. The method does not require a healthy counterpart or a reference sample for the analysis and can be therefore applied also to transcriptional data generated from cell lines. In our implementation the tool can use gene-expression data from microarray experiments, as well as data generated by RNASeq technologies. CONCLUSIONS: The method was implemented as a publicly available, user-friendly tool called KAOS (Kinase Automatic Outliers Search). The tool enables the automatic execution of iterative searches for the identification of extreme outliers and for the graphical visualization of the results. Filters can be applied to select the most significant outliers. The performance of the tool was evaluated using a synthetic dataset and compared to state-of-the-art tools. KAOS performs particularly well in detecting genes that are overexpressed in few samples or when an extreme outlier stands out on a high variable expression background. To validate the method on real case studies, we used publicly available tumor cell line microarray data, and we were able to identify genes which are known to be overexpressed in specific samples, as well as novel ones.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

BACKGROUND: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. METHODS: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. RESULTS: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. CONCLUSIONS: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors.

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.

Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

Discovery of drug mode of action and drug repositioning from transcriptional responses.

A bottleneck in drug discovery is the identification of the molecular targets of a compound (mode of action, MoA) and of its off-target effects. Previous approaches to elucidate drug MoA include analysis of chemical structures, transcriptional responses following treatment, and text mining. Methods based on transcriptional responses require the least amount of information and can be quickly applied to new compounds. Available methods are inefficient and are not able to support network pharmacology. We developed an automatic and robust approach that exploits similarity in gene expression profiles following drug treatment, across multiple cell lines and dosages, to predict similarities in drug effect and MoA. We constructed a "drug network" of 1,302 nodes (drugs) and 41,047 edges (indicating similarities between pair of drugs). We applied network theory, partitioning drugs into groups of densely interconnected nodes (i.e., communities). These communities are significantly enriched for compounds with similar MoA, or acting on the same pathway, and can be used to identify the compound-targeted biological pathways. New compounds can be integrated into the network to predict their therapeutic and off-target effects. Using this network, we correctly predicted the MoA for nine anticancer compounds, and we were able to discover an unreported effect for a well-known drug. We verified an unexpected similarity between cyclin-dependent kinase 2 inhibitors and Topoisomerase inhibitors. We discovered that Fasudil (a Rho-kinase inhibitor) might be "repositioned" as an enhancer of cellular autophagy, potentially applicable to several neurodegenerative disorders. Our approach was implemented in a tool (Mode of Action by NeTwoRk Analysis, MANTRA, http://mantra.tigem.it).

Novel Thienoduocarmycin-Trastuzumab ADC Demonstrates Strong Antitumor Efficacy with Favorable Safety Profile in Preclinical Studies.

New antibodies-drug conjugate (ADC) payloads overcoming chemoresistance and killing also poorly proliferating tumors at well-tolerated doses are much desired. Duocarmycins are a well-known class of highly potent cytotoxic agents, with DNA minor groove-binding and alkylation properties, active also in chemoresistant tumors. Although different duocarmycin derivatives have been used during the years as payloads for ADC production, unfavorable physicochemical properties impaired the production of ADCs with optimal features. Optimization of the toxin to balance reactivity and stability features and best linker selection allowed us to develop the novel duocarmycin-like payload-linker NMS-P945 suitable for conjugation to mAbs with reproducible drug-antibody ratio (DAR) >3.5. When conjugated to trastuzumab, it generated an ADC with good internalization properties, ability to induce bystander effect and immunogenic cell death. Moreover, it showed strong target-driven activity in cells and cytotoxic activity superior to trastuzumab deruxtecan tested, in parallel, in cell lines with HER2 expression. High in vivo efficacy with cured mice at well-tolerated doses in HER2-driven models was also observed. A developed pharmacokinetic/pharmacodynamic (PK/PD) model based on efficacy in mice and cynomolgus monkey PK data, predicted tumor regression in patients upon administration of 2 doses of trastuzumab-NMS-P945-ADC at 0.5 mg/kg. Thus, considering the superior physicochemical features for ADC production and preclinical results obtained with the model trastuzumab ADC, including bystander effect, immunogenic cell death and activity in chemoresistant tumors, NMS-P945 represents a highly effective, innovative payload for the creation of novel, next-generation ADCs.

The generation of purinome-targeted libraries as a means to diversify ATP-mimetic chemical classes for lead finding.

The generation of novel chemotypes in support of our oncology research projects expanded in recent years from a canonical design of kinase-targeted compound libraries to a broader interpretation of purinome-targeted libraries (PTL) addressing the specificity of cancer relevant targets such as kinases and ATPases. Successful screening of structurally diverse ATP-binding targets requires compound libraries covering multiple design elements, which may include phosphate surrogate moieties in ATPase inhibitors or far reaching lipophilic residues stabilizing inactive kinase conformations. Here, we exemplify the design and preparation of drug-like combinatorial libraries and report significantly enhanced screening performance on purinomic targets. We compared overall hit rates of PTL with a simultaneously tested unbiased collection of 200,000 compounds and found consistent superiority of the targeted libraries in all cases. We also analyzed the performance of the largest targeted libraries in comparison with each other and often found striking differences in how a specific target responds to various chemotypes and to whole collections.

Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.

Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Mining potentially actionable kinase gene fusions in cancer cell lines with the KuNG FU database.

Inhibition of kinase gene fusions (KGFs) has proven successful in cancer treatment and continues to represent an attractive research area, due to kinase druggability and clinical validation. Indeed, literature and public databases report a remarkable number of KGFs as potential drug targets, often identified by in vitro characterization of tumor cell line models and confirmed also in clinical samples. However, KGF molecular and experimental information can sometimes be sparse and partially overlapping, suggesting the need for a specific annotation database of KGFs, conveniently condensing all the molecular details that can support targeted drug development pipelines and diagnostic approaches. Here, we describe KuNG FU (KiNase Gene FUsion), a manually curated database collecting detailed annotations on KGFs that were identified and experimentally validated in human cancer cell lines from multiple sources, exclusively focusing on in-frame KGF events retaining an intact kinase domain, representing potentially active driver kinase targets. To our knowledge, KuNG FU represents to date the largest freely accessible homogeneous and curated database of kinase gene fusions in cell line models.

Crystal structure of the T315I Abl mutant in complex with the aurora kinases inhibitor PHA-739358.

Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine.