Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics.

Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model.

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.

A restricted role for TYK2 catalytic activity in human cytokine responses revealed by novel TYK2-selective inhibitors.

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including type I IFNs, IL-6, IL-10, IL-12, and IL-23. Extensive studies of mice that lack TYK2 expression indicate that the IFN-α, IL-12, and IL-23 pathways, but not the IL-6 or IL-10 pathways, are compromised. In contrast, there have been few studies of the role of TYK2 in primary human cells. A genetic mutation at the tyk2 locus that results in a lack of TYK2 protein in a single human patient has been linked to defects in the IFN-α, IL-6, IL-10, IL-12, and IL-23 pathways, suggesting a broad role for TYK2 protein in human cytokine responses. In this article, we have used a panel of novel potent TYK2 small-molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic activity in cytokine pathways in primary human cells. Our results indicate that the biological processes that require TYK2 catalytic function in humans are restricted to the IL-12 and IL-23 pathways, and suggest that inhibition of TYK2 catalytic activity may be an efficacious approach for the treatment of select autoimmune diseases without broad immunosuppression.

Design and evaluation of novel 8-oxo-pyridopyrimidine Jak1/2 inhibitors.

A highly ligand efficient, novel 8-oxo-pyridopyrimidine containing inhibitor of Jak1 and Jak2 isoforms with a pyridone moiety as the hinge-binding motif was discovered. Structure-based design strategies were applied to significantly improve enzyme potency and the polarity of the molecule was adjusted to gain cellular activity. The crystal structures of two representative inhibitors bound to Jak1 were obtained to enable SAR exploration.

2-Amino-[1,2,4]triazolo[1,5-a]pyridines as JAK2 inhibitors.

The advancement of a series of ligand efficient 2-amino-[1,2,4]triazolo[1,5-a]pyridines, initially identified from high-throughput screening, to a JAK2 inhibitor with pharmacodynamic activity in a mouse xenograft model is disclosed.

Structure-based discovery of C-2 substituted imidazo-pyrrolopyridine JAK1 inhibitors with improved selectivity over JAK2.

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

Structure Based Design of Potent Selective Inhibitors of Protein Kinase D1 (PKD1).

We previously disclosed a series of type I 1/2 inhibitors of NF-κB inducing kinase (NIK). Inhibition of NIK by these compounds was found to be strongly dependent on the inclusion and absolute stereochemistry of a propargyl tertiary alcohol as it forms critical hydrogen bonds (H-bonds) with NIK. We report that inhibition of protein kinase D1 (PKD1) by this class of compounds is not dependent on H-bond interactions of this tertiary alcohol. This feature was leveraged in the design of highly selective inhibitors of PKD1 that no longer inhibit NIK. A structure-based hypothesis based on the position and flexibility of the α-C-helix of PKD1 vs NIK is presented.

Getting to know your neighbours; a new mechanism for cell intercalation.

As an embryo gastrulates or makes neural tissue it shortens across the dorso-ventral axis and extends dramatically along the perpendicular, antero-posterior axis, resulting in the embryo doubling in length. This process is known as convergent extension and it is so powerful in remodelling tissue that it is used time and again during development. New research in Drosophila melanogaster and other model organisms is shedding fresh light on how it happens.

Lung-restricted inhibition of Janus kinase 1 is effective in rodent models of asthma.

Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as interleukin-4 (IL-4), IL-5, IL-9, and IL-13. Additional evidence predicts pathogenic roles for IL-6 and type I and type II interferons. Because each of these cytokines depends on Janus kinase 1 (JAK1) for signal transduction, and because many of the asthma-related effects of these cytokines manifest in the lung, we hypothesized that lung-restricted JAK1 inhibition may confer therapeutic benefit. To test this idea, we synthesized iJak-381, an inhalable small molecule specifically designed for local JAK1 inhibition in the lung. In pharmacodynamic models, iJak-381 suppressed signal transducer and activator of transcription 6 activation by IL-13. Furthermore, iJak-381 suppressed ovalbumin-induced lung inflammation in both murine and guinea pig asthma models and improved allergen-induced airway hyperresponsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect on neutrophil-driven inflammation compared to systemic corticosteroid administration. The inhibitor iJak-381 reduced lung pathology, without affecting systemic Jak1 activity in rodents. Our data show that local inhibition of Jak1 in the lung can suppress lung inflammation without systemic Jak inhibition in rodents, suggesting that this strategy might be effective for treating asthma.

NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

A Rho GTPase signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation.

A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation . It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. We show here that in vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Galpha(12/13) family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding.

A deformation gradient decomposition method for the analysis of the mechanics of morphogenesis.

A new finite element model is proposed for the analysis of the mechanical aspects of morphogenesis and tested on the biologically well studied gastrulation phenomenon, in particular ventral furrow invagination of the Drosophila melanogaster embryo. A set of mechanisms are introduced in the numerical model, which lead to the observed deformed shapes. We split the total deformation into two parts: an imposed active deformation, and an elastic deformation superimposed onto the latter. The active deformation simulates the effects of apical constriction and apico-basal elongation. These mechanisms are associated with known gene expressions and so in this way we attempt to bridge the well explored signalling pathways, and their associated phenotypes in a mechanical model. While the former have been studied in depth, much less can be said about the forces they produce and the mechanisms involved. From the numerical results, we are able to test different plausible mechanical hypotheses that generate the necessary folding observed in the invagination process. In particular, we conclude that only certain ratios between both modes (apical constriction and apico-basal elongation) can successfully reproduce the invagination process. The model also supports the idea that this invagination requires the contribution of several mechanisms, and that their redundancy provides the necessary robustness.

Tetrahydroindazoles as Interleukin-2 Inducible T-Cell Kinase Inhibitors. Part II. Second-Generation Analogues with Enhanced Potency, Selectivity, and Pharmacodynamic Modulation in Vivo.

The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response.

Interleukin-2 (IL-2)-inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.

Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress.

Facioscapulohumeral muscular dystrophy is an autosomal dominant disorder resulting from an unusual genetic mechanism. The mutation, a deletion of 3.3 kb subtelomeric repeats, appears to disrupt the regional regulation of 4q35 g ene expression. The specific gene(s)responsible for facioscapulohumeral muscular dystrophy have not been identified. However, the 'vacuolar/necrotic' phenotype exhibited by facioscapulohumeral muscular dystrophy myoblasts suggests that aberrant gene expression occurs early in facioscapulohumeral muscular dystrophy muscle development. In order to test this hypothesis, global gene expression profiling and in vitro characterization of facioscapulohumeral muscular dystrophy and control myoblasts were carried out. Genes involved in several cellular processes such as oxidative stress were found to be dysregulated. In vitro studies confirmed this susceptibility to oxidative stress, as proliferative stage facioscapulohumeral muscular dystrophy myoblasts exhibit greatly reduced viability when exposed to the oxidative stressor paraquat. This effect was not seen in either normal or disease control myoblasts, or in any of the cell lines upon differentiation to multinucleated myotubes. Immunocytochemical studies of the cyclin dependent kinase inhibitor p21 demonstrated increased expression in facioscapulohumeral muscular dystrophy myoblasts, suggesting an early cell cycle arrest. Another process distinguishing facioscapulohumeral muscular dystrophy from controls involves the transcription of extracellular matrix components. Expression of elastin, decorin, lumican and the extracellular matrix remodeling factor TIMP3 were reduced in facioscapulohumeral muscular dystrophy myoblasts. These studies suggest that facioscapulohumeral muscular dystrophy muscular dystrophy results from a defect in early myogenesis, manifested as increased susceptibility to oxidative stress, morphological aberrations and early cell cycle arrest.

Evaluation of a whitening gel designed to accelerate whitening.

A clinical trial was conducted with 25 subjects to evaluate the effects and safety of Zoom! Take-Home whitening gel, a 6% hydrogen peroxide gel for vital tooth bleaching. Tooth bleaching was accomplished using a tray system overnight for 6 nights. Over the 6 nights, a significant change (from darker to lighter) was seen in tooth shades as demonstrated by 3 assessment methods: VITA Shade Value Oriented Guide, Trubyte Bioform Color Ordered Shade Guide scoring system, and the Chroma Meter CR-321 assessments. VITA Shade scores showed a mean change of -6.49 shades (P = .0001) from baseline to day 4 and a -7.72 shades (P = .0001) from baseline to day 7. The Trubyte scores showed a mean change of -9.31 shades (P = .0001) from baseline to day 4 and a -10.77 shades (P = .0001) from baseline to day 7. The Chroma Meter was used to measure tooth color. Analysis of Chroma Meter data showed a significant change in color (delta E), 13.82 mean score change (P = .0001) from baseline to day 4 and a 7.25 mean score change (P = .0001) from baseline to day 7. At day 4 minor tooth sensitivity was reported, and all tooth sensitivity resolved within a few days after treatment.

Lead optimization of a 4-aminopyridine benzamide scaffold to identify potent, selective, and orally bioavailable TYK2 inhibitors.

Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.

Lead identification of novel and selective TYK2 inhibitors.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.