A carboxy terminal BMP/TGF-ß binding site in secreted phosphoprotein 24 kD independently affects BMP-2 activity.

Secreted phosphoprotein 24 kD (spp24) is a bone matrix protein isolated during attempts to identify osteogenic proteins. It is not osteogenic but performs other important roles in the regulation of bone metabolism, at least in part, by binding to and affecting the activity of members of the BMP/TGF-ß family of cytokines. Spp24 exists in a number of forms that preserve the N-terminus and are truncated at the C-terminus. The hypothesized cytokine binding domain is present within the cystatin domain which is preserved in all of the N-terminal products. In this report, we describe a C-terminal fragment that is distinct from the cystatin domain and which independently binds to BMP-2 and TGF-ß. This fragment inhibited BMP-2 activity in an ectopic bone forming assay. A shorter C-terminal product did not inhibit BMP-2 activity but improved bone quality induced by BMP-2 and produced increased calcium deposition outside of bone. Spp24 has been used to develop several potential therapeutic proteins. These results provide more information on the function of spp24 and provide other materials that can be exploited for clinical interventions.

YC-1 binding to the ß subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the ß subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 µM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 µM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 µM) than to the isolated heme domain (Kd = 0.2 µM for Manduca ß H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 µM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

Tandem Mass Tagging Based Identification of Proteome Signatures for Reductive Stress Cardiomyopathy.

Nuclear factor erythroid 2-related factor 2 (NRF2), a redox sensor, is vital for cellular redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2-TG) exhibit reductive stress (RS). In this study, we identified novel protein signature for RS-induced cardiomyopathy using Tandem Mass Tag (TMT) proteomic analysis in heart tissues of TG (CaNrf2-TG) mice at 6-7 months of age. A total of 1,105 proteins were extracted from 22,544 spectra. About 560 proteins were differentially expressed in TG vs. NTg hearts, indicating a global impact of RS on the myocardial proteome. Over 32 proteins were significantly altered in response to RS -20 were upregulated and 12 were downregulated in the hearts of TG vs. NTg mice, suggesting that these proteins could be putative signatures of RS. Scaffold analysis revealed a clear distinction between TG vs. NTg hearts. The majority of the differentially expressed proteins (DEPs) that were significantly altered in RS mice were found to be involved in stress related pathways such as antioxidants, NADPH, protein quality control, etc. Interestingly, proteins that were involved in mitochondrial respiration, lipophagy and cardiac rhythm were dramatically decreased in TG hearts. Of note, we identified the glutathione family of proteins as the significantly changed subset of the proteome in TG heart. Surprisingly, our comparative analysis of NGS based transcriptome and TMT-proteome indicated that ~50% of the altered proteins in TG myocardium was found to be negatively correlated with their transcript levels. In association with the altered proteome the TG mice displayed pathological cardiac remodeling.

Not so Dangerous After All? Venom Composition and Potency of the Pholcid (Daddy Long-Leg) Spider Physocyclus mexicanus.

Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1-17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.

Discovery of quinolinediones exhibiting a heat shock response and angiogenesis inhibition.

A series of substituted quinoline-5,8-diones were synthesized and evaluated as inhibitors of the chaperone protein Hsp90 using two assays: competition for binding to C-terminal ATP-binding site and competition for binding to N-terminal ATP-binding site. In addition, the ability of the compounds to induce the heat shock response was determined using a reporter fibroblast cell line. Of all the compounds assayed, only 6-aziridinyl-2-biphenylquinoline-5,8-dione induced a heat shock response and did so without interacting at the ATP binding sites of Hsp90. COMPARE analysis was carried out on quinoline-5,8-diones active in the National Cancer Institute's 60-cell line screen with the goal of discovering quinoline-5,8-dione structures that interact with other cellular targets (molecular targets) important for cancer chemotherapy. COMPARE analysis led to the discovery of a combretastatin-like quinoline-5,8-dione structure that, in fact, inhibited angiogenesis.

Radiologic findings in taxane induced colitis.

Ischemic colitis in breast cancer patients being treated with taxane-based chemotherapy, which may lead to serious morbidities and even death, has recently been defined as a clinical entity. The purpose of this retrospective study was to evaluate the computed tomography (CT) findings in taxane-related colitis and determine their clinical relevance. CT scans of 41 patients at risk for taxane colitis were reviewed retrospectively for bowel and peritoneal abnormalities. Morphological findings were analyzed and correlated with clinical, pathological, and endoscopic findings. CT scans in 10 of the 41 patients showed a definitely abnormal colon with a thickened wall or distended with fluid, signs that are suggestive of colitis, in the context of the clinical picture. Radiographic changes in patients with taxane colitis are not specific but, in the appropriate context, can suggest the correct diagnosis and guide the patient's management.

Phase II evaluation of gemcitabine monotherapy for cutaneous T-cell lymphoma.

PURPOSE: The purpose of this study was to investigate safety and efficacy of gemcitabine monotherapy for cutaneous T-cell lymphoma (CTCL). PATIENTS AND METHODS: Twenty-five patients with CTCL on a phase II open-label trial and 8 patients off study received intravenous gemcitabine (1000 mg/m2) on day 1, 8, and 15 for > or = 6 cycles. Physicians' global assessment was based on body surface area involvement in skin, measurement of lymph nodes, and blood by flow cytometry. RESULTS: Two patients with CD30+ anaplastic large T-cell lymphoma and 31 with mycosis fungoides (stage IB [T2, n = 2], stage IIA [T2, n = 1], stage IIB [T3, n = 13], stage IVA [T3 N3, n = 3; T4b2, n = 2; T4b2 N3, n = 2], and stage IVB [T4b2 N1, n = 6; T4 N3b2 M1, n = 1; T3 N3 M1, n = 1]) had received a median of 5 previous therapies (range, 1-13 therapies). Responses were seen in 17 of 25 (68%) study patients (2 complete responses [8%]) and 4 of 8 patients (1 complete response) off protocol. Seven of 13 patients with mycosis fungoides (T3) responded, 10 had tumor burden reductions, and 8 of 11 patients with Sezary syndrome responded. Gemcitabine was well tolerated. Myelosuppression (n = 14; grade 3, n = 8), hemolytic uremic syndrome (in 2 elderly patients with Sezary syndrome), pulmonary embolism (n = 2), and 1 episode each of congestive heart failure, acute myocardial infarction, and stable angina were observed. Increased hepatic transaminases (n = 4), mucositis (n = 3), lethargy (n = 7), fever (n = 8), cutaneous hyperpigmentation (n = 6), infusion-related maculopapular rash (n = 1), and radiation recall (n = 1) were also seen. CONCLUSION: Gemcitabine is an effective monotherapy with a 68% overall response rate in patients with advanced, heavily pretreated CTCL.

A Phase I study of docetaxel plus cyclophosphamide in solid tumors followed by a Phase II study as first-line therapy in metastatic breast cancer.

PURPOSE: In Phase I, the purpose was to determine the maximum tolerated dose and pharmacokinetics of docetaxel plus cyclophosphamide (DC) with and without granulocyte colony-stimulating factor in the treatment of patients with solid tumors. For Phase II, the purpose was to determine the safety and efficacy of this combination as first-line treatment in patients with metastatic breast cancer (MBC). EXPERIMENTAL DESIGN: In Phase I (45 patients), docetaxel was escalated from 60 mg/m(2) to 85 mg/m(2), and cyclophosphamide from 600 mg/m(2) to 800 mg/m(2). Pharmacokinetic evaluation of docetaxel was performed in 19 patients with MBC. In Phase II (34 patients), patients received cyclophosphamide (600 mg/m(2)) followed by docetaxel (75 mg/m(2)), i.v. RESULTS: In Phase I, the dose-limiting toxicity was neutropenia-related events. The maximum tolerated dose for DC was 75 mg/m(2)/700 mg/m(2) in solid tumor patients treated previously and 75 mg/m(2)/800 mg/m(2) for patients not treated previously for MBC. Dose escalation of docetaxel >75 mg/m(2) was not tolerated, despite prophylactic granulocyte colony-stimulating factor treatment. In Phase II, 71% of patients received prior anthracycline therapy. Neutropenic fever requiring i.v. antibiotics occurred in 6 patients (19%). One patient had grade 3 neuropathy. There was no cardiotoxicity. The overall Phase II intent-to-treat objective response rate was 65% (complete responses, 12%). The median overall survival was 22 months, and the median time to progression was 6 months. CONCLUSIONS: DC combination therapy is an active regimen with acceptable toxicity and is appropriate regardless of prior anthracycline therapy. In view of the high activity and lack of cardiotoxicity, this combination warrants additional investigation in early stage breast cancer and in combination with trastuzumab.

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans.

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

Plectreurys tristis venome: A proteomic and transcriptomic analysis.

Spider venoms are complex cocktails rich in peptides, proteins and organic molecules that collectively act to immobilize prey. Venoms of the primitive hunting spider, Plectreurys tristis, have numerous neurotoxic peptides called "plectoxins" (PLTX), a unique acylpolyamine called bis(agmatine)oxalamide, and larger unidentified protein components. These spiders also have unconventional multi-lobed venom glands. Inspired by these unusual characteristics and their phylogenetic position as Haplogynes, we have partially characterized the venome of P. tristis using combined transcriptomic and proteomic methods. With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups. The venom also contains proteins that are homologs of astacin metalloproteases that, combined with venom peptides, make up 94% of components detected in crude venom, while the remaining 6% is a single undescribed protein with unknown function. Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts. These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks. Finally, we show that crude venom has neurotoxic effects and an effective paralytic dose on crickets of 3.3µg/gm.

The suppressor of AAC2 Lethality SAL1 modulates sensitivity of heterologously expressed artemia ADP/ATP carrier to bongkrekate in yeast.

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner.

Search for Hsp90 inhibitors with potential anticancer activity: isolation and SAR studies of radicicol and monocillin I from two plant-associated fungi of the Sonoran desert.

In an effort to discover small molecule inhibitors of Hsp90, we have screened over 500 EtOAc extracts of Sonoran desert plant-associated fungi using a two-stage strategy consisting of a primary cell-based heat shock induction assay (HSIA) followed by a secondary biochemical luciferase refolding assay (LRA). Bioassay-guided fractionation of extracts active in these assays derived from Chaetomium chiversii and Paraphaeosphaeria quadriseptata furnished the Hsp90 inhibitors radicicol (1) and monocillin I (2), respectively. In SAR studies, 1, 2, and their analogues, 3-16, were evaluated in these assays, and the antiproliferative activity of compounds active in both assays was determined using the breast cancer cell line MCF-7. Radicicol and monocillin I were also evaluated in a solid-phase competition assay for their ability to bind Hsp90 and to deplete cellular levels of two known Hsp90 client proteins with relevance to breast cancer, estrogen receptor (ER), and the type 1 insulin-like growth factor receptor (IGF-1R). Some inferences on SAR were made considering the crystal structure of the N-terminus of yeast Hsp90 bound to 1 and the observed biological activities of 1-16. Isolation of radicicol and monocillin I in this study provides evidence that we have developed an effective strategy for discovering natural product-based Hsp90 inhibitors with potential anticancer activity.

Hsp90 inhibitors deplete key anti-apoptotic proteins in pediatric solid tumor cells and demonstrate synergistic anticancer activity with cisplatin.

Drugs that inhibit the function of heat shock protein 90 (Hsp90) are of interest in the treatment of pediatric cancers because these agents deplete the cellular levels of signaling molecules that are important for the growth and survival of many childhood tumors. To generate preclinical data in anticipation of clinical trials of Hsp90 inhibitors in children, we evaluated the effects of the Hsp90 inhibitor geldanamycin (GA) alone and in combination with cis-platinum (II)-diamine dichloride (cisplatin) in pediatric tumor cells. Immunoblotting demonstrated depletion of the Hsp90 client proteins AKT and the type 1 insulin-like growth factor receptor (IGF1R) in a panel of pediatric tumor cell lines after exposure to GA. Drug exposure also led to a dramatic decrease in cell survival/proliferation in MYCN-amplified and non-amplified neuroblastoma cells. Moderate inhibition of survival/proliferation was observed in RB-deficient and wild-type osteosarcoma cells. Treatment of neuroblastoma and osteosarcoma cell lines with GA in combination with cisplatin resulted in greater than additive inhibition of survival/proliferation based on median dose analysis. Exposure to this drug combination also resulted in a marked increase in nuclear fragmentation as assessed by terminal deoxynucleotide transferase-mediated UTP nick end labeling (TUNEL) analysis. Combined exposure also abrogated the ability of GA to induce a cytoprotective heat shock response and resulted in Hsp90 adduct formation. Our findings suggest that Hsp90 inhibitors may prove useful either alone or as a component of multi-drug regimens in the treatment of neuroblastoma and osteosarcoma.

Design of quinolinedione-based geldanamycin analogues.

Quinoline-5,8-dione-based compounds were designed from the structure of the geldanamycin-bound Hsp-90 active site. The active site model predicted that aromatic substituents should be present at the 2-position, to take advantage of a hydrophobic pocket, and amino substituents should be present at the 6- or 7-position. COMPARE analysis revealed that the LC(50) profile of 2-phenyl-6-(2-chloroethylamino)quinoline-5,8-dione has the highest geldanamycin-like activity (0.74 correlation coefficient).