17 research outputs found
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FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.
Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance
Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.
Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2
Single-cell phenotypic characterization of Staphylococcus aureus with fluorescent triazole urea activity-based probes.
Phenotypically distinct cellular (sub)populations are clinically relevant for virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Here, we introduce fluorescent activity-based probes as chemical tools for single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose chemical probe labeling of enzymatic activities as a generalizable method for phenotyping of bacterial cells at the population and single-cell level
The partial-thickness rotator cuff tear: is acromioplasty without repair sufficient ?
Contains fulltext :
mmubn000001_02748713x.pdf (publisher's version ) (Open Access)Promotor : A. Manning
cum laudeXVI, 329 p
Electrostatic and hydrogen bonding interactions between charged and polar amino acid side chains.
<p>(A) PEA-15 DED in the free form (PDB ID 2LS7). (B) L-arginine (CSD Refcode TAQBIY). (C) Full-length PEA-15 complexed with T185E ERK2 (PDB ID 4IZ5). (D) PEA-15 DED complexed with unphosphorylated ERK2 (PDB ID 4IZ7). (E) PEA-15 DED complexed with dual phosphorylated ERK2 (PDB ID 4IZA). (F) MC159 protein DED1 (PDB ID 2BBR). (G) MC159 protein DED2 (PDB ID 2BBR). The six helices, α1–α6, are labeled on each DED structure, with residues forming hydrogen bonds represented as a stick model (cyan color with pink labels), and heavy atom distances (in Å) shown between the two residues. Residues from ERK2 (C–E) are colored in orange with green labels.</p
Comparison of experimentally-measured and predicted residual dipolar couplings (RDCs) of PEA-15 death effector domain (DED, residues 1–90).
<p>Experimental RDCs (red) were measured for the PEA-15 in the free (A, B) and ERK2-bound (C–F) forms in two alignment media: filamentous bacteriophage pf1 (A, C, E) and non-ionic C<sub>12</sub>E<sub>5</sub>/<i>n</i>-hexanol (B, D, F). Predicted RDCs (blue) were calculated from (A–D) the NMR model structure of free PEA-15 (2LS7) or (E, F) the ERK2-bound form of PEA-15 (4IZ7). The positions of the alpha-helices are indicated by pink bars. The free-form PEA-15 displays excellent agreement between the experimental and predicted RDC values throughout the DED sequence, while the ERK2-bound PEA-15 RDCs only reasonably agree in helices α1, α5, and α6 for both 2LS7 and 4IZ7, indicating a significant reorientation of helices α2, α3, and α4 in the PEA-15/ERK2 complex.</p
Distribution of distances between nitrogen and oxygen atoms involved in N-H…O hydrogen bonds in small molecule structures containing intermolecular carboxylate-guanidine-carboxylate (charge-triad mimic) motifs surveyed in the Cambridge Structural Database (CSD).
<p>A total of 109 structures that possess the charge-triad mimics were extracted from the CSD and hydrogen bonding patterns analyzed to generate the histogram. Only intermolecular hydrogen bonds formed between COO<sup>−</sup>…H-N<sup>+</sup>-H…<sup>−</sup>OOC were counted in the histogram.</p
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Erythropoietic Protoporphyria: Phase 2 Clinical Trial Results Evaluating the Safety and Effectiveness of Dersimelagon (MT-7117), an Oral MC1R Agonist
Introduction Erythropoietic Protoporphyria (EPP) or X-Linked Protoporphyria (XLP) are inborn errors of heme biosynthesis caused by the abnormal accumulation of erythrocyte protoporphyrin IX (ePPIX). These protoporphyrias are characterized by excruciating painful attacks on prolonged sunlight exposure. Before the onset of phototoxic pain, patients experience characteristic prodromal symptoms which serves as a warning signal to avoid further sun exposure. Of note, patients with higher ePPIX levels report shorter times to symptoms compared to patients with lower median ePPIX levels (JAMA Derm, 2017; 153). Here we report the safety and effectiveness of Dersimelagon (MT-7117), a novel, orally-administered, small molecule, selective melanocortin-1 receptor (MC1R) agonist that increases skin melanin without sun exposure and is being developed to increase light tolerance in EPP/XLP patients. Results of the primary, secondary efficacy endpoints, and post-hoc subgroup analyses evaluating effects of baseline ePPIX levels on treatment response will be presented. Methods The MT-7117-A01 (ENDEAVOR) study was a Phase 2, multi-center, randomized, placebo-controlled study with a 16-week double-blind treatment period. A total of 102 EPP/XLP patients were randomized to 3 groups: placebo once daily (QD) (n=35 [31 EPP/4 XLP]), MT-7117 100 mg QD (n=33 [31 EPP/2 XLP]), and MT-7117 300 mg QD (n=34 [31 EPP/3 XLP]). Results of the primary endpoint, increase in the average daily time (min) to first prodromal symptom during sunlight exposure, and secondary endpoints including 1) average daily duration of sunlight exposure without prodromal symptoms, 2) total number of sunlight exposure episodes with prodromal symptoms, and 3) total number of pain events, and post-hoc subgroup analyses evaluating baseline median ePPIX levels are presented here. Safety and tolerability were also assessed. Results There was a significant improvement in average daily time (>50 min) to first prodromal symptom [the primary endpoint] associated with sunlight exposure in subjects treated with MT-7117 100 mg (p=0.008) or 300 mg (p=0.003) compared to placebo at Week 16. Multiple secondary endpoints supported primary endpoint. There was a significant increase in average daily minutes of sunlight exposure without prodromal symptoms at Week 16 in 100 mg (p=0.009) and 300 mg (p=0.004) treated subjects compared to placebo, with an increased average exposure time without prodromal symptoms of ~50 min in MT-7117 subjects at both doses compared to placebo. There was also a 40% reduction in the total number of sunlight exposure episodes with prodromal symptoms in 100 mg (p=0.019) and 300 mg (p=0.006) subjects compared to placebo. There was a significant decrease in the total number of pain events reported in 100 mg (p=0.027, 60% reduction) and 300 mg (p=0.028, 50% reduction) subjects compared to placebo. The post-hoc subgroup analysis evaluating the effects of baseline ePPIX levels in subjects grouped based on the baseline median ePPIX level of 1981 µg/dL. There was a statistically significant increase in average daily time to first prodromal symptom in the subgroup with ePPIX levels ≥1981 µg/dL receiving 100 mg (p=0.020) and 300 mg (p=0.003) compared to placebo. A trend to beneficial effect was also observed for the subgroup of subjects with ePPIX levels <1981 µg/dL receiving 100 mg (p=0.180) and 300 mg (p=0.216) compared to placebo. MT-7117 had an acceptable safety and tolerability profile. The most common treatment-related treatment emergent adverse reactions were nausea (27.9%), ephelides (23.5%), and skin hyperpigmentation (20.6%). Conclusion The results of the primary efficacy endpoint and multiple secondary endpoints in this Phase 2 study indicate that the oral, MC1R agonist dersimelagon was efficacious after 16 weeks of treatment in increasing symptom-free light exposure in patients with EPP or XLP at doses of 100 and 300 mg QD and showed an acceptable safety and tolerability profile. The post-hoc analyses showed more profound and statistically significant efficacy at both doses for the subgroup with higher baseline median ePPIX levels. Disclosures Balwani: Alnylam Pharmaceuticals: Consultancy, Honoraria, Research Funding; Recordati Rare Diseases: Consultancy, Honoraria, Other: Disease information video recording. Anderson:Alnylam Pahrmaceuticals: Consultancy; Recordati Rare Diseases: Consultancy; Mitsubishi Tanabe Pharma: Consultancy