Discovery and optimization of 4-pyrazolyl-2-aminopyrimidine derivatives as potent spleen tyrosine kinase (SYK) inhibitors.

Spleen tyrosine kinase (Syk) is a key signal transduction mediator of the B cell receptor (BCR) signaling pathway. Abnormal BCR signaling plays a key role in initiation and development of B-cell-derived hematological malignancies, therefore, Syk represents a potential target for inhibiting the BCR signaling resulting in a therapeutic effect in these cancers. Herein, we describe a novel series of SYK inhibitors with 4-(3'-pyrazolyl)-2-amino-pyrimidine scaffold. Extensive study of structure-activity relationships led to the identification of 1 (NMS-0963), a highly potent Syk inhibitor (IC50 = 3 nM) endowed with high selectivity within a panel of tested kinases and high antiproliferative activity in SYK-dependent BaF3-TEL/SYK cells and in other BCR-dependent hematological tumor cell lines. Additionally, 1 effectively inhibited Syk phosphorylation and downstream signaling mediators of the BCR in treated cells. In in vivo pharmacokinetics studies, 1, displayed good pharmacokinetics properties, with linear exposure with dose and excellent oral bioavailability. These findings suggest that 1 is a promising new Syk inhibitor for treating BCR-dependent hematological cancers.

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.

Effects of a short period of postural training on postural stability and vestibulospinal reflexes.

The effects of postural training on postural stability and vestibulospinal reflexes (VSRs) were investigated in normal subjects. A period (23 minutes) of repeated episodes (n = 10, 50 seconds) of unipedal stance elicited a progressive reduction of the area covered by centre of pressure (CoP) displacement, of average CoP displacement along the X and Y axes and of CoP velocity observed in this challenging postural task. All these changes were correlated to each other with the only exception of those in X and Y CoP displacement. Moreover, they were larger in the subjects showing higher initial instability in unipedal stance, suggesting that they were triggered by the modulation of sensory afferents signalling body sway. No changes in bipedal stance occurred soon and 1 hour after this period of postural training, while a reduction of CoP displacement was apparent after 24 hours, possibly due to a beneficial effect of overnight sleep on postural learning. The same period of postural training also reduced the CoP displacement elicited by electrical vestibular stimulation (EVS) along the X axis up to 24 hours following the training end. No significant changes in postural parameters of bipedal stance and VSRs could be observed in control experiments where subjects were tested at identical time points without performing the postural training. Therefore, postural training led to a stricter control of CoP displacement, possibly acting through the cerebellum by enhancing feedforward mechanisms of postural stability and by depressing the VSR, the most important reflex mechanism involved in balance maintenance under challenging conditions.

EV20/NMS-P945, a Novel Thienoindole Based Antibody-Drug Conjugate Targeting HER-3 for Solid Tumors.

HER-3 is becoming an attractive target for antibody-drug conjugate (ADC)-based therapy. Indeed, this receptor and its ligands are found to be overexpressed in several malignancies, and re-activation of its downstream signaling axis is known to play a critical role in modulating the sensitivity of targeted therapeutics in different tumors. In this study, we generated a novel ADC named EV20/NMS-P945 by coupling the anti-HER-3 antibody EV20 with a duocarmycin-like derivative, the thienoindole (TEI) NMS-P528, a DNA minor groove alkylating agent through a peptidic cleavable linker. This ADC showed target-dependent cytotoxic activity in vitro on several tumor cell lines and therapeutic activity in mouse xenograft tumor models, including those originating from pancreatic, prostatic, head and neck, gastric and ovarian cancer cells and melanoma. Pharmacokinetics and toxicological studies in monkeys demonstrated that this ADC possesses a favorable terminal half-life and stability and it is well tolerated. These data support further EV20/NMS-P945 clinical development as a therapeutic agent against HER-3-expressing malignancies.

Multisensory Integration Is Modulated by Hypnotizability.

This study investigated multisensory integration in 29 medium-to-high (mid-highs) and 24 low-to-medium (mid-lows) hypnotizable individuals, classified according to the Stanford Hypnotic Susceptibility Scale, Form A. Participants completed a simultaneity judgment (SJ) task, where an auditory and a visual stimulus were presented in close proximity to their body in a range of 11 stimulus onset asynchronies. Results show that mid-highs were prone to judge audiovisual stimuli as simultaneous over a wider range of time intervals between sensory stimuli, as expressed by a broader temporal binding window, when the visual stimulus precedes the auditory one. No significant difference was observed for response times. Findings indicate a role of hypnotizability in multisensory integration likely due to the highs' cerebellar peculiarities and/or sensory modality preference.

Postural effects of interoceptive imagery as a function of hypnotizability.

The aim of the study was to investigate the effects of pleasant and unpleasant interoceptive imagery on postural control in participants with different hypnotizability, interoceptive sensibility and ability of imagery. Forty-one healthy individuals classified as high (highs), medium (mediums) and low hypnotizables (lows) according to the Stanford Hypnotic Susceptibility Scale, form A (SHSS, A) were characterized for Interoceptive Sensitivity (IS) through the Multisensory Assessment of Interoceptive Awareness (MAIA) and for organic mental imagery (ORG) through Betts' questionnaire. The experimental session included baseline closed eyes conditions preceding tasks of pleasant (P) and unpleasant imagery (U) and a neutral cognitive task (NT) while standing on a stabilometric platform. Subjective reports of vividness and pleasantness/unpleasantness of mental images were collected. Postural variables, ECG and pneumogram were acquired. Highs exhibited greater vividness of imagery than mediums/lows and larger Area of the Centre of Pressure (CoP), while mediums and lows decreased it during all tasks with respect to baseline conditions. Significant differences moderated by IS and ORG were found between highs and lows in the CoP Area. In all groups significant task related differences in the CoP Area were moderated only by IS. Cardiovascular variables were similar in the three groups, but differed among tasks and were influenced by IS and ORG. Our findings extend earlier observations on the role of hypnotisability in the postural correlates of sensorimotor imagery to interoceptive imagery, support the hypothesis that interoceptive sensibility moderates postural control and cardiorespiratory variables during interoceptive imagery, and confirm earlier reports of the absence of hypnotisability-related modulation of cardiorespiratory variables during emotional tasks.

Nonlinear indentation of single human erythrocytes under application of a localized mechanical force.

Despite the accepted notion that erythrocytes are uniquely deformable cells, the apparent Young's modulus values reported in the literature do not differ so much from those of other cells. We devised to measure the local deformability of living immobilized human erythrocytes at a low force, in contact-free mode, using an application of Scanning Ion Conductance Microscopy (SICM) previously developed in our laboratory. Reversible indentations were induced by forces of up to few hundreds pN. The indentation did not grow linearly with the force. The apparent Young's modulus varied from 0.2 to 1.5 kPa applying forces from 20 to 500 pN on a cell surface area of about 0.2 µm2, exhibiting a progressive stiffening at increasing force. Control measurements showed that A549 cells exhibit a constant value of the apparent Young's modulus (about 2 kPa) for forces up to about 800 pN. These findings show that SICM is a suitable tool to investigate cell mechanical properties, when forces in the range of tens of pN are required, in the absence of mechanical contact between probe and sample. The nonlinear deformation of the erythrocyte has to be taken into account in modeling the complex regulation mechanism of the microvascular beds.

Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

Asymmetric Tactile Foot Stimulation: How Postural Studies May Suggest New Views of Hypnotizability.

Earlier studies have shown hypnotizability-related postural effects of visual suppression and of leg and neck proprioceptive alteration. This study completes this investigation by demonstrating the postural effects of asymmetric tactile foot stimulation in standing participants with different hypnotizability scores. During this stimulation, body sway changed in medium-to-high more than in low-to-medium hypnotizable participants. Findings support the view that high hypnotizability is associated with higher vulnerability of posture to sensory alteration; together with earlier results, they suggest a role of the cerebellum in the observed hypnotizability-related differences and prompt investigation of cerebral structures and factors potentially responsible for both the cognitive and physiological aspects of hypnotizability.

Blink reflex in subjects with different hypnotizability: New findings for an old debate.

Hypnotizability is associated with attentional characteristics whose neurophysiological bases are still under debate. Aim of the study was the assessment of possible hypnotizability-related differences in blink reflex (BR) which has a nociceptive component, is sensitive to attentional-emotional traits and states and is modulated by the brain dopamine content. In 10 high (highs) and 10 low hypnotizable participants (lows) BR was induced by electrical nociceptive stimulation of the right supraorbital nerve in the absence (noW) and in the presence of a visual cue preceding the electrical stimulation by 0.1ms (W01) and by 1ms (W1). The studied variables were: the amplitude of BR components (R1, R2, R3), the amplitude of the quick change (TO) of heart rate ("turbulence") induced by stimulation and its recovery slope (TS), the role of the Behavioral Inhibition/Activation System (BIS/BAS) in the variability BR and cardiac turbulence. Repeated measures ANOVA did not show any significant difference between highs and lows in blink reflex. TO indicated stimulation related HR increase in highs and decrease in lows, TS was larger in highs. BIS and BAS accounted for the warning effects on the BR amplitude and modulated the hypnotizability and warning effects on TO and TS. Findings do not support dopamine based hypnotizability-related attentional abilities. In contrast, they indicate that hypnotizability modulates the short-lasting cardiac response to electrical nociceptive stimulation.

Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

Discovery of Entrectinib: A New 3-Aminoindazole As a Potent Anaplastic Lymphoma Kinase (ALK), c-ros Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer Therapy.

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.

Revisiting the association between hypnotisability and blink rate.

Blink rate (BR), which is considered an index of the dopaminergic tone, has been studied in 41 subjects with high (highs), medium (mediums) and low (lows) hypnotisability scores in resting conditions. It has been found higher in highs; relaxation (indicated by skin conductance), anxiety and the proneness to absorption in tasks (assessed by questionnaires) were not responsible for the observed difference. In contrast, the BR difference did not survive controlling for mind wandering (MW questionnaire) whose variability could account for contrasting earlier reports, although no significant hypnotisability-related difference has been observed in MW scores. Findings do not allow to exclude that mechanisms other than dopaminergic ones may be involved in the observed difference in BR. In particular, we suggest that one of the mechanisms possibly sustaining the highs' higher BR may be a reduced cerebellar inhibition. In fact, cerebellar impairment is associated with higher BR and several studies of sensorimotor integration indicate different cerebellar controls in the highs' and lows' behavior.

Two-photon polymerization of sub-micrometric patterned surfaces: investigation of cell-substrate interactions and improved differentiation of neuron-like cells.

Direct Laser Writing (DLW) is an innovative tool that allows the photofabrication of high resolution 3D structures, which can be successfully exploited for the study of the physical interactions between cells and substrates. In this work, we focused our attention on the topographical effects of submicrometric patterned surfaces fabricated via DLW on neuronal cell behavior. In particular, we designed, prepared, and characterized substrates based on aligned ridges for the promotion of axonal outgrowth and guidance. We demonstrated that both rat PC12 neuron-like cells and human SH-SY5Y derived neurons differentiate on parallel 2.5 µm spaced submicrometric ridges, being characterized by strongly aligned and significantly longer neurites with respect to those differentiated on flat control substrates, or on more spaced (5 and 10 µm) ridges. Furthermore, we detected an increased molecular differentiation toward neurons of the SH-SY5Y cells when grown on the submicrometric patterned substrates. Finally, we observed that the axons can exert forces able of bending the ridges, and we indirectly estimated the order of magnitude of these forces thanks to scanning probe techniques. Collectively, we showed as submicrometric structures fabricated by DLW can be used as a useful tool for the study of the axon mechanobiology.

Measuring the elastic properties of living cells through the analysis of current-displacement curves in scanning ion conductance microscopy.

Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe-sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current-displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young's modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.

Weak hydrostatic forces in far-scanning ion conductance microscopy used to guide neuronal growth cones.

Scanning ion conductance microscopy (SICM) is currently used for high resolution topographic imaging of living cells. Recently, it has been also employed as a tool to deliver stimuli to the cells. In this work we have investigated the mechanical interaction occurring between the pipette tip and the sample during SICM operation. For the purpose, we have built a setup combining SICM with atomic force microscopy (AFM), where the AFM cantilever replaces the sample. Our data indicate that, operating in far-scanning mode with current decrease values below 2%, no force can be detected, provided that the level of the electrolyte filling the pipette is equal to that determined by the capillary tension. A filling level different from this value determines a hydrostatic pressure, a flux through the pipette tip and detectable forces, even in far-scanning mode. The absolute value of these forces depends on the pipette tip size. Therefore, a possible pitfall when using SICM for cell imaging is to imply zero-force working conditions. However the hydrostatic forces can be exploited in order to deliver weak mechanical stimuli and guide neuronal growth cones. Evidences of the effectiveness of this approach are herein given.

Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding.

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.