To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized.
Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0 Å resolution and were consistent with the primitive trigonal space group P212121.
IgCAM3 domain; myosin light-chain kinase 1
Class II major histocompatability molecules are the primary susceptibility locus for many autoimmune disorders including type 1 diabetes. Human DQ8 and I-Ag7, in the non-obese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We utilized an in silico molecular docking program to screen a large “drug-like” chemical library to define small molecules capable of occupying specific structural pockets along the I-Ag7 binding groove with the objective of influencing presentation of the autoantigen insulin B:9–23 to T cells. In this study we demonstrate using both murine and human cells that small molecules can enhance or inhibit specific T cell receptor (TCR) signaling in the presence of cognate target peptides based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing response to peptide. At nanomolar concentrations, the inhibitory molecules block insulin B:9–23 peptide, endogenous insulin, and islet stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses with potentially therapeutic applications.
Deregulation of insulin-like growth factor-1 receptor (IGF-1R) and focal adhesion kinase (FAK) signaling pathways plays an important role in cancer cell proliferation and metastasis. In pancreatic cancer cells, the crosstalk and compensatory mechanisms between these two pathways reduce the efficacy of the treatments that target only one of the pathways. Ablation of IGF-1R signaling by siRNA showed minimal effects on the survival and growth of pancreatic cancer cells. An increased activity of FAK pathway was seen in these cells after IGF-1R knockdown. Further inhibition of FAK pathway using Y15 significantly decreased cell survival, adhesion, and promoted apoptosis. The combination of Y15 treatment and IGF-1R knockdown also showed significant antitumor effect in vivo. The current study demonstrates the importance of dual inhibition of both these signaling pathways as a novel strategy to decrease both in vitro and in vivo growth of human pancreatic cancer.
FAK; IGF-1R; pancreatic cancer
The development of lupus pathogenesis results from the integration of susceptibility and resistance genes. We have used a chronic graft-versus-host disease (cGVHD) model to characterize a suppressive locus at the telomeric end of the NZM2410-derived Sle2 susceptibility locus, which we named Sle2c2. cGVHD is induced normally in Sle2c2-expressing mice, but it is not sustained. The analysis of mixed bone marrow chimeras revealed that cGVHD resistance was eliminated by non-B non-T hematopoietic cells expressing the B6 allele, suggesting that resistance is mediated by this same cell type. Furthermore, Sle2c2 expression was associated with an increased number and activation of the CD11b+ GR-1+ subset of granulocytes before and in the early stage of cGVHD induction. We have mapped the Sle2c2 critical interval to a 6-Mb region that contains the Cfs3r gene, which encodes for the G-CSFR, and its NZM2410 allele carries a nonsynonymous mutation. The G-CSFR–G-CSF pathway has been previously implicated in the regulation of GVHD, and our functional data on Sle2c2 suppression suggest a novel regulation of T cell-induced systemic autoimmunity through myeloid-derived suppressor cells. The validation of Csf3r as the causative gene for Sle2c2 and the further characterization of the Sle2c2 MDSCs promise to unveil new mechanisms by which lupus pathogenesis is regulated.
The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.
Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER) pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase β (Pol-β) and identified a potent anti-Pol-β compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ), by blocking BER. First, we determined the specificity of NSC-124854 for Pol-β by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR)-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-β and blocked Pol-β-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression.
Characterization of immune receptors found in phylogenetically disparate species at the genetic, structural and functional levels has provided unique insight into the evolutionary acquisition of immune function. The roles of variable- and intermediate-type immunoglobulin (Ig) domains in direct recognition of ligands and other functions are far wider than previously anticipated. Common mechanisms of multigene family diversification and expansion as well as unique adaptations that relate to function continue to provide unique insight into the numerous patterns, processes and complex interactions that regulate the host response to infectious challenge.
immunoglobulin domain; functional diversity; evolutionary variation; chimeric function; adaptive and innate immunity
The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase β (Pol-β) and identified a potent small molecular weight inhibitor (SMI), NSC-666715. We determined the specificity of this SMI for Pol-β by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. The binding specificity of NSC-666715 with Pol-β was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug, Temozolomide (TMZ), to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-β activity. It blocked Pol-β-directed SN- and LP-BER without affecting the activity of APE1, Fen1 and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-β and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug-resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the “proof-of-concept” for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer.
Adenomatous polyposis coli; DNA polymerase β; small molecular weight inhibitors; colorectal cancer; chemotherapeutic intervention
The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assay using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (aa 127–243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the biding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.
FAK is a tyrosine kinase that functions as a key orchestrator of signals leading to invasion and metastasis. Since FAK interacts directly with a number of critical proteins involved in survival signaling in tumor cells, we hypothesized that targeting a key protein-protein interface with drug-like small molecules was a feasible strategy for inhibiting tumor growth. In this study, we targeted the protein-protein interface between FAK and VEGFR-3 and identified compound C4 (chloropyramine hydrochloride) as a drug capable of 1) inhibiting the biochemical function of VEGFR-3 and FAK, 2) inhibiting proliferation of a diverse set of cancer cell types in vitro, and 3) reducing tumor growth in vivo. Chloropyramine hydrochloride reduced tumor growth as a single agent, while concomitant administration with doxorubicin had a pronounced synergistic effect. Our data demonstrate that the FAK-VEGFR-3 interaction can be targeted by small drug-like molecules and this interaction can provide the basis for highly-specific novel cancer therapeutics.
An interaction between the B2 subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments is required for osteoclast bone resorption. An atomic homology model of the actin binding site on B2 was generated and molecular docking simulations were performed. Enoxacin, a fluoroquinolone antibiotic, was identified and in vitro testing demonstrated that enoxacin blocked binding between purified B2 and microfilaments. Enoxacin dose dependently reduced the number of osteoclasts differentiating in mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, as well as markers of osteoclast activity, and the number of resorption lacunae formed on bone slices. Enoxacin inhibited osteoclast formation at concentrations where osteoblast formation was not altered. In summary, enoxacin is a novel small molecule inhibitor of osteoclast bone resorption that acts by an unique mechanism and is therefore an attractive lead molecule for the development of a new class of antiosteoclastic agents.
X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Å resolution.
X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Å resolution and reduced to the primitive orthorhombic lattice. A single molecule was predicted to be present in the asymmetric unit based on the Matthews coefficient. The data were phased using molecular-replacement methods using an existing model of the FAK FAT domain. All structures of human focal adhesion kinase FAT domains solved to date have been solved in a C-centered orthorhombic space group.
focal adhesion kinase; targeting domain
Rationale: It has been proposed that an activated renin angiotensin system (RAS) causes an imbalance between the vasoconstrictive and vasodilator mechanisms involving the pulmonary circulation leading to the development of pulmonary hypertension (PH). Recent studies have indicated that angiotensin-converting enzyme 2 (ACE2), a member of the vasoprotective axis of the RAS, plays a regulatory role in lung pathophysiology, including pulmonary fibrosis and acute lung disease. Based on these observations, we propose the hypothesis that activation of endogenous ACE2 can shift the balance from the vasoconstrictive, proliferative axis (ACE-Ang II-AT1R) to the vasoprotective axis [ACE2-Ang-(1–7)-Mas] of the RAS, resulting in the prevention of PH.
Objectives: We have taken advantage of a recently discovered synthetic activator of ACE2, XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), to study its effects on monocrotaline-induced PH in rats to support this hypothesis.
Methods: The cardiopulmonary effects of XNT were evaluated in monocrotaline-induced PH rat model.
Measurements and Main Results: A single subcutaneous treatment of monocrotaline in rats resulted in elevated right ventricular systolic pressure, right ventricular hypertrophy, increased pulmonary vessel wall thickness, and interstitial fibrosis. These changes were associated with increases in the mRNA levels of renin, ACE, angiotensinogen, AT1 receptors, and proinflammatory cytokines. All these features of PH were prevented in these monocrotaline-treated rats by chronic treatment with XNT. In addition, XNT caused an increase in the antiinflammatory cytokine, IL-10.
Conclusions: These observations provide conceptual support that activation of ACE2 by a small molecule can be a therapeutically relevant approach for treating and controlling PH.
renin angiotensin system; angiotensin-converting enzyme 2; pulmonary heart disease.
Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors. We developed a novel cancer-therapy approach, targeting the main autophosphorylation site of FAK, Y397 by computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database. More than 140,000 small molecule compounds were docked into the N-terminal domain of the FAK crystal structure in 100 different orientations that identified 35 compounds. One compound 14 (1,2,4,5-Benzenetetraamine tetrahydrochloride) significantly decreased viability in most of the cells to the levels equal or higher than control FAK inhibitor, 1a (2-[5-Chloro-2-[2-methoxy-4-(4-morpholinyl)phenylamino]pyrimidin-4-ylamino]-N-methylbenzamide; TAE226) from Novartis, Inc. The compound 14 specifically and directly blocked phosphorylation of Y397-FAK in a dose- and time-dependent manner. It increased cell detachment and inhibited cell adhesion in a dose-dependent manner. Furthermore, 14 effectively caused breast tumor regression in vivo. Thus, targeting the Y397 site of FAK with 14 inhibitor can be effectively used in cancer therapy.
Focal Adhesion Kinase; Y397 site; autophosphorylation; tumor; inhibitor
A highly diversified novel immune-type receptor from catfish, NITR10, was crystallized to reveal novel mechanisms of immune recognition.
X-ray diffraction data from crystals of a novel immune-type receptor (NITR10 from the catfish Ictalurus punctatus) were collected to 1.65 Å resolution and reduced to the primitive hexagonal lattice. Native and selenomethionine derivatives of NITR10 crystallized under different conditions yielded P3121 crystals. SeMet NITR10 was phased to a correlation coefficient of 0.77 by SAD methods and experimental electron-density maps were calculated to 1.65 Å. Five NITR10 molecules are predicted to be present in the asymmetric unit based on the Matthews coefficient.
immune-type receptors; NITR10; Ictalurus punctatus
Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating/inhibitory receptors that has been identified in bony fish. In this study, we characterize the structure of an activating NITR that is expressed by a cytotoxic NK-like cell line and specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by x-ray crystallography reveal: (1) the V-type domains of NITRs form homodimers resembling heterodimers formed by rearranging antigen binding receptors and (2) both subunits of NITR dimers form ligand-binding surfaces in CDR1 that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK-type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immune responses.
Jak2 tyrosine kinase is essential for animal development and hyper-kinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high throughput program called DOCK, to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3) bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner, but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect of Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase.
Small Molecule Inhibitor; Jak2; Myeloproliferative Disorders
The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.
Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actin-binding activity has been demonstrated in the B-subunit of yeast. A conserved “profilin-like” domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the “profilin-like” domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the “housekeeping” functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida
albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.
Osteoclast; vacuolar ATPase; actin; microfilaments; enoxacin; cancer; Candida.