Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets.
View Article and Find Full Text PDFEnzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors.
View Article and Find Full Text PDFBivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets.
View Article and Find Full Text PDFIntegrins α4β1/ α9β1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4β1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought.
View Article and Find Full Text PDFPolyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model.
View Article and Find Full Text PDFAdvancement of stem cell-based treatment will involve next-generation approaches to enhance therapeutic efficacy which is often modest, particularly in the context of myocardial regenerative therapy. Our group has previously demonstrated the beneficial effect of genetic modification of cardiac stem cells with Pim-1 kinase overexpression to rejuvenate aged cells as well as potentiate myocardial repair. Despite these encouraging findings, concerns were raised regarding potential for oncogenic risk associated with Pim-1 kinase overexpression.
View Article and Find Full Text PDFWe propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [Anal. Biochem.
View Article and Find Full Text PDFPim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge.
View Article and Find Full Text PDFSpleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells.
View Article and Find Full Text PDFStem cell-specific proteins and regulatory pathways that determine self-renewal and differentiation have become of fundamental importance in understanding regenerative and reparative processes in the myocardium. One such regulatory protein, named nucleostemin, has been studied in the context of stem cells and several cancer cell lines, where expression is associated with proliferation and maintenance of a primitive cellular phenotype. We find nucleostemin is present in young myocardium and is also induced following cardiomyopathic injury.
View Article and Find Full Text PDFActivation of cyclin-dependent kinase 1 (Cdk1) has been linked to cell death of postmitotic neurons in brain development and disease. We found that Cdk1 phosphorylated the transcription factor FOXO1 at Ser249 in vitro and in vivo. The phosphorylation of FOXO1 at Ser249 disrupted FOXO1 binding with 14-3-3 proteins and thereby promoted the nuclear accumulation of FOXO1 and stimulated FOXO1-dependent transcription, leading to cell death in neurons.
View Article and Find Full Text PDFCumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population.
View Article and Find Full Text PDFThe serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans.
View Article and Find Full Text PDFActivation of Akt is associated with enhanced cell cycling and cellular proliferation in nonmyocytes, but this effect of nuclear Akt accumulation has not been explored in the context of the myocardium. Cardiac-specific expression of nuclear-targeted Akt (Akt/nuc) in transgenics prolongs postnatal cell cycling as evidenced by increased numbers of Ki67+ cardiomyocytes at 2 to 3 weeks after birth. Similarly, nuclear-targeting of Akt promotes expansion of the presumptive cardiac progenitor cell population as assessed by immunolabeling for c-kit in combination with myocyte-specific markers Nkx 2.
View Article and Find Full Text PDFContinuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)-induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin-GIT1 binding and promoting the localization of a GIT1-PIX-PAK signaling module near the leading edge.
View Article and Find Full Text PDFProtein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases.
View Article and Find Full Text PDFThis study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation.
View Article and Find Full Text PDFCell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect.
View Article and Find Full Text PDFPurpose: c-MET is believed to be an attractive receptor target for molecular therapeutic inhibition. TPR-MET, a constitutively active oncogenic variant of MET, serves as excellent model for testing c-MET inhibitors. Here, we characterized a small molecule c-MET inhibitor, PHA665752, and tested its cooperation with the mammalian target of rapamycin inhibitor as potential targeted therapy.
View Article and Find Full Text PDFNon-small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues.
View Article and Find Full Text PDFIntegrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion.
View Article and Find Full Text PDFEndothelial cells lining the vasculature have close cell-cell associations that maintain separation of the blood fluid compartment from surrounding tissues. Permeability is regulated by a variety of growth factors and cytokines and plays a role in numerous physiological and pathological processes. We examined a potential role for the p21-activated kinase (PAK) in the regulation of vascular permeability.
View Article and Find Full Text PDFAltered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering.
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