MyMD-1 Improves Health Span and Prolongs Life Span in Old Mice: A Noninferiority Study to Rapamycin.

Aging and age-related diseases represent a compelling therapeutic goal for senolytics and drugs targeting inflammatory or metabolic pathways. We compared MyMD-1, a synthetic derivative of the alkaloid myosmine capable of suppressing TNF-α production, to rapamycin, the best characterized drug endowed with antiaging properties. In vivo, a longitudinal cohort of 54 C57BL/6 mice, 19-month-old at the start, was randomized to receive MyMD-1, high-dose (126 ppm) rapamycin, or low-dose (14 ppm) rapamycin plus metformin. Each treatment arm included 18 mice (10 females and 8 males) and was followed for 16 months or until death. Life span was significantly longer in MyMD-1 than rapamycin (p = .019 vs high-dose and .01 vs low-dose) in a Cox survival model that accounted for sex and serum levels of IL-6, TNF-α, and IL-17A. MyMD-1 also improved several health span characteristics, resulting in milder body weight loss, greater muscle strength, and slower progression to frailty. In vitro, MyMD-1 and rapamycin were compared using a panel of 12 human primary cell systems (BioMAP Diversity PLUS), where a total of 148 biomarkers were measured. MyMD-1 possessed antiproliferative, anti-inflammatory, and antifibrotic properties. Many were shared with rapamycin, but MyMD-1 was more active in the inhibition of proinflammatory and profibrotic biomarkers. Overall, MyMD-1 emerges as a new compound that, even when begun at an advanced age, induces beneficial effects on health and life span by modulating inflammation and tissue remodeling.

A tubulin binding molecule drives differentiation of acute myeloid leukemia cells.

Despite much progress in developing better drugs, many patients with acute myeloid leukemia (AML) still die within a year of diagnosis. This is partly because it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Overcoming this block should allow for the identification of therapies that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify compounds that stimulate differentiation in genetically diverse AML cell lines. Lead compounds were shown to decrease tumor burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a function for tubulin disruptors in causing differentiation of AML cells.

Comparative phenotypic profiling of the JAK2 inhibitors ruxolitinib, fedratinib, momelotinib, and pacritinib reveals distinct mechanistic signatures.

Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling is critical to multiple cellular processes, including survival, differentiation, and proliferation. JAK-STAT signaling dysregulation has been noted in inflammatory disorders, and aberrant JAK2 pathway activation has been implicated in myelofibrosis and polycythemia vera. Moreover, 4 therapeutic JAK2 inhibitors (ruxolitinib, fedratinib, momelotinib, and pacritinib) have either been approved or are in advanced clinical development for myelofibrosis. Although all inhibit JAK2, reports indicate that they also inhibit other kinases. Profiling based solely on in vitro potencies is insufficient to predict the observed clinical effects. To provide further translational insights into clinical outcomes, we compared phenotypic biomarker profiles of ruxolitinib, fedratinib, momelotinib, and pacritinib in the BioMAP® Diversity PLUS panel of 12 human primary cell systems designed to recapitulate key aspects of tissue and disease states. Biomarker activity profiles that represent mechanistic signatures for each agent were compared with each other and a database of reference benchmark profiles. At clinically relevant concentrations, these agents had distinct biomarker impacts indicating diverse mechanistic signatures, suggesting divergent clinical effects for each agent. They disparately modulated inflammatory cytokine production and immune function. At clinically relevant concentrations, ruxolitinib had the broadest scope of activities across all 12 cellular systems, whereas pacritinib was more specific for the BT system (modelling T cell-dependent B cell activation) and exhibited the strongest inhibition of sIL-17A, sIL-2, and sIL-6. All 4 agents were antiproliferative to B cells, but ruxolitinib and momelotinib were also antiproliferative to T cells. These differential activities likely reflect distinct secondary pharmacology for these agents known primarily as JAK2 inhibitors. The phenotypic analysis reported herein represents key data on distinct modes-of-action that may provide insights on clinical outcomes reported for these agents. Such translational findings may also inform the development of next-generation molecules with improved efficacy and safety.

Discriminating phenotypic signatures identified for tocilizumab, adalimumab, and tofacitinib monotherapy and their combinations with methotrexate.

BACKGROUND: Clinical trials have shown combinations of anti-tumor necrosis factor biologicals plus methotrexate (MTX) are more effective treatments for rheumatoid arthritis than biological monotherapies, based, in part, on the assumption that MTX reduces the immunogenicity of biologicals. However, co-treatment with the anti-interleukin-6 receptor-alpha antibody tocilizumab (TCZ) and MTX does not demonstrate the same level of incremental benefit over TCZ monotherapy. Using the human primary cell based BioMAP phenotypic profiling platform, we investigated the impact of TCZ, adalimumab (ADA), and the small molecule drug tofacitinib (TOF), alone and in combination with MTX, on translational biomarkers that could indicate unique pharmacodynamic interactions outside those of reduced immunogenicity. METHODS: TCZ, ADA, and TOF, alone and in combination with MTX, were profiled in BioMAP systems at concentrations close to clinical exposure levels: TCZ, 200 µg/ml; TOF1, 1.1 µM; TOF2, 0.12 µM; MTX, 10 µM. Changes in biomarkers were evaluated by statistical methods to determine whether combinations differed from the individual agents. RESULTS: Although the BioMAP activity profile for TCZ + MTX was not significantly different from that for TCZ alone, profiles for ADA + MTX and TOF1 + MTX or TOF2 + MTX had a greater number of statistically significant different activities (P < 0.01) than did agents profiled individually. CONCLUSIONS: These data support the comparable efficacy of TCZ as monotherapy and as combination therapy and suggest that TOF, like ADA, may be more beneficial in combination with MTX. Taking an orthogonal approach to directly compare monotherapy and combination therapies indicates that MTX contributes to the efficacy of some, but not all, RA therapies and can be affected by factors additional to reduced immunogenicity.

Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation.

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2-/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2-/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2-/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2-/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators.

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy.

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

Elucidating mechanisms of toxicity using phenotypic data from primary human cell systems--a chemical biology approach for thrombosis-related side effects.

Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.

A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation.

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.

RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC(50) = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC(50) = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

NF-kappaB/Rel regulates inhibitory and excitatory neuronal function and synaptic plasticity.

Changes in synaptic plasticity required for memory formation are dynamically regulated through opposing excitatory and inhibitory neurotransmissions. To explore the potential contribution of NF-kappaB/Rel to these processes, we generated transgenic mice conditionally expressing a potent NF-kappaB/Rel inhibitor termed IkappaBalpha superrepressor (IkappaBalpha-SR). Using the prion promoter-enhancer, IkappaBalpha-SR is robustly expressed in inhibitory GABAergic interneurons and, at lower levels, in excitatory neurons but not in glia. This neuronal pattern of IkappaBalpha-SR expression leads to decreased expression of glutamate decarboxylase 65 (GAD65), the enzyme required for synthesis of the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) in GABAergic interneurons. IkappaBalpha-SR expression also results in diminished basal GluR1 levels and impaired synaptic strength (input/output function), both of which are fully restored following activity-based task learning. Consistent with diminished GAD65-derived inhibitory tone and enhanced excitatory firing, IkappaBalpha-SR+ mice exhibit increased late-phase long-term potentiation, hyperactivity, seizures, increased exploratory activity, and enhanced spatial learning and memory. IkappaBalpha-SR+ neurons also express higher levels of the activity-regulated, cytoskeleton-associated (Arc) protein, consistent with neuronal hyperexcitability. These findings suggest that NF-kappaB/Rel transcription factors act as pivotal regulators of activity-dependent inhibitory and excitatory neuronal function regulating synaptic plasticity and memory.

Human T-cell lymphotropic virus type 1 tax induction of biologically Active NF-kappaB requires IkappaB kinase-1-mediated phosphorylation of RelA/p65.

Activation of the NF-kappaB/Rel family of transcription factors proceeds through a catalytic complex containing IkappaB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-kappaB transcriptional program. In IKK1(-/-) murine embryonic fibroblasts (MEFs), Tax normally induced early NF-kappaB activation events. However, NF-kappaB induced by Tax in these IKK1(-/-) cells was functionally impaired. In IKK1(-/-) (but not wild-type) MEFs, Tax failed to activate several different kappaB reporter constructs or to induce the endogenous IkappaBalpha gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1(-/-) cells. Furthermore, reconstitution of IKK1(-/-) cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-kappaB transactivation. We further found that the defect in NF-kappaB action in IKK1(-/-) cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-kappaB at Ser(529) and Ser(536). Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser(536) was required for a complete response to Tax expression, whereas phosphorylation of Ser(529) appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-kappaB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IkappaBalpha degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-kappaB by promoting RelA/p65 phosphorylation.