Abstract Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (−)−epigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC50 of 14 μmol/L). EGCG abrogated anchorage-independent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598–605)
Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21→A). We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue. The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.
The TLQP-21 neuropeptide has been implicated in functions as diverse as lipolysis, neurodegeneration and metabolism, thus suggesting an important role in several human diseases. Three binding targets have been proposed for TLQP-21: C3aR1, gC1qR and HSPA8. The aim of this review is to critically evaluate the molecular identity of the TLQP-21 receptor and the proposed multi-receptor mechanism of action. Several studies confirm a critical role for C3aR1 in TLQP-21 biological activity and a largely conserved mode of binding, receptor activation and signaling with C3a, its first-identified endogenous ligand. Conversely, data supporting a role of gC1qR and HSPA8 in TLQP-21 activity remain limited, with no signal transduction pathways being described. Overall, C3aR1 is the only receptor for which a necessary and sufficient role in TLQP-21 activity has been confirmed thus far. This conclusion calls into question the validity of a multi-receptor mechanism of action for TLQP-21 and should inform future studies.
Musterbildung mit Proteinen: Ein Protein, das mit einer Azidmarkierung selektiv gekennzeichnet war, wurde mit alkinfunktionalisierter DNA umgesetzt. Die Protein-DNA-Konjugate wurden genutzt, um eine mit DNA (blau) aggregierte Proteinnanostruktur (weiß) herzustellen. Die Methode könnte die Erzeugung von Protein-DNA-Hybridstrukturen mit nützlichen strukturellen und funktionellen Eigenschaften vereinfachen.
Introduction: Alpha-1 adrenergic receptor (α1-AR) antagonists (α1-blockers) are efficacious antihypertensives, but ALLHAT found that doxazosin (Dzn) doubled the risk of heart failure (HF). We previously demonstrated that α1-ARs, specifically the α1A-AR, protect the heart, explaining the worse outcomes with Dzn in ALLHAT. Interestingly, α1-ARs localize to and signal at the nucleus in cardiac myocytes (CM), whereas α1-ARs localize to the sarcolemma in smooth muscle (SM). Hypothesis: We hypothesize that a membrane impermeant α1-blocker would inhibit α1-ARs in SM, reducing blood pressure, but preserve protective α1-signaling in CMs, preventing HF. Methods: We developed an α1A-AR homology model to define the structural basis of α1-blocker binding and modified existing α1-blocker structure to reduce cardiotoxicity. Using this model, we designed 30 compounds, synthesized 9 in gram quantities, and tested 2 compounds. Results and Conclusions: Binding assays identified 2 novel high affinity α1-ligands; α1705 (IC50 = 5.2 [CI:3.5,7.6] nM, n=3) and α1707 (5.8 [CI:4.2,8.1] nM, n=3) (Dzn: 7.7 [CI:6.3,9.2] nM, n=6). Uptake assays in cultured CM indicated uptake of Prazosin (10 μM Prz: 230 nM in CMs detected by mass spectrometry), whereas α1705 and α1707 were not detected. In HEK cells expressing α1A-AR at the cell surface, Prz, α1705, and α1707 (all 2 μM) inhibited Ca2+ transients (90%, 76%, 88%) induced by the α1A agonist A61603 (20 μM). In CM, A61603 increased ERK phosphorylation (1.55±0.04 fold, n=7, P<0.0001), a survival signal, which was blocked by Prz (1.21±0.06 fold, n=7, P=NS), but not α1705 (1.72±0.13 fold, n=7, P<0.001). Further, A61603 (20 μM) inhibited H202-induced CM death (-17.4±7.6%, n=5), which was reversed by Prz (30.9±15.4%, n=5, P=0.035), but not α1705 (-0.94±11.6%, n=5, P=NS). In DOCA-salt hypertensive mice, infusion of Prz or α1705 (2 mg/kg/d, 3 d) lowered systolic pressure (Cont: +18.9 mmHg, n=1; Prz: 6.4±3.2 mmHg, n=3, α1705: -6.1±5.6 mmHg, n=3), and while Prz was detected in CMs following 4 d, α1705 was not. In conclusion, we have developed a novel membrane impermeant α1-blocker with reduced cardiotoxicity that could improve hypertension outcomes, especially in patients suffering from chronic kidney disease or benign prostatic hyperplasia.
We describe a visualization system designed for interactive study of proteins in the field of computational biology. Our system incorporates multiple, custom, three-dimensional and two-dimensional linked views of the proteins. We take advantage of modern commodity graphics cards, which are typically designed for games rather than scientific visualization applications, to provide instantaneous linking between views and three-dimensional interactivity on standard personal computers. Furthermore, we anticipate the usefulness of game techniques such as bump maps and skinning for scientific applications.
<div>AbstractPurpose:<p>Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months.</p>Experimental Design:<p>This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14<sup>+</sup> cells. GMP-grade peptide was further validated for activity.</p>Results:<p>We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors.</p>Conclusions:<p>These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.</p></div>
The bacterial metallo-β-lactamases (MBLs) catalyze the inactivation of β-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar β-lactamase inhibitor of Verona-integron-encoded metallo-β-lactamase 2 (VIM-2), capable of rescuing β-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-β-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.
