Structure guided development of potent piperazine-derived hydroxamic acid inhibitors targeting falcilysin.

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria and is estimated to kill 400,000 people a year. The parasite infects and replicates in host red blood cells (RBCs), where it expresses an array of proteases to carry out multiple essential processes. We are investigating the function of falcilysin (FLN), a protease known to be required for parasite development in the RBC. We previously developed a piperazine-based hydroxamic acid scaffold to generate the first inhibitors of FLN, and the current study reports the optimization of the lead compound from that series. A range of substituents were tested at the N1 and N4 positions of the piperazine core, and inhibitors with significantly improved potency against purified FLN and cultured P. falciparum were identified. Computational studies were also performed to understand the mode of binding for these compounds, and predicted a binding model consistent with the biochemical data and the distinctive SAR observed at both the N1 and N4 positions.

Development of piperazine-based hydroxamic acid inhibitors against falcilysin, an essential malarial protease.

The human parasite Plasmodium falciparum kills an estimated 445,000 people a year, with the most fatalities occurring in African children. Previous studies identified falcilysin (FLN) as a malarial metalloprotease essential for parasite development in the human host. Despite its essentiality, the biological roles of this protease are not well understood. Here we describe the optimization of a piperazine-based hydroxamic acid scaffold to develop the first reported inhibitors of FLN. Inhibitors were tested against cultured parasites, and parasiticidal activity correlated with potency against FLN. This suggests these compounds kill P. falciparum by blocking FLN, and that FLN is a druggable target. These compounds represent an important step towards validating FLN as a therapeutic target and towards the development of chemical tools to investigate the function of this protease.

Probing the orientation of inhibitor and epoxy-eicosatrienoic acid binding in the active site of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is an important therapeutic target of many diseases, such as chronic obstructive pulmonary disease (COPD) and diabetic neuropathic pain. It acts by hydrolyzing and thus regulating specific bioactive long chain polyunsaturated fatty acid epoxides (lcPUFA), like epoxyeicosatrienoic acids (EETs). To better predict which epoxides could be hydrolyzed by sEH, one needs to dissect the important factors and structural requirements that govern the binding of the substrates to sEH. This knowledge allows further exploration of the physiological role played by sEH. Unfortunately, a crystal structure of sEH with a substrate bound has not yet been reported. In this report, new photoaffinity mimics of a sEH inhibitor and EET regioisomers were prepared and used in combination with peptide sequencing and computational modeling, to identify the binding orientation of different regioisomers and enantiomers of EETs into the catalytic cavity of sEH. Results indicate that the stereochemistry of the epoxide plays a crucial role in dictating the binding orientation of the substrate.

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin.

Heparan sulfate differences in rheumatoid arthritis versus healthy sera.

Heparan sulfate (HS) is a complex and highly variable polysaccharide, expressed ubiquitously on the cell surface as HS proteoglycans (HSPGs), and found in the extracellular matrix as free HS fragments. Its heterogeneity due to various acetylation and sulfation patterns endows a multitude of functions. In animal tissues, HS interacts with a wide range of proteins to mediate numerous biological activities; given its multiple roles in inflammation processes, characterization of HS in human serum has significant potential for elucidating disease mechanisms. Historically, investigation of HS was limited by its low concentration in human serum, together with the complexity of the serum matrix. In this study, we used a modified mass spectrometry method to examine HS disaccharide profiles in the serum of 50 women with rheumatoid arthritis (RA), and compared our results to 51 sera from healthy women. Using various purification methods and online LC-MS/MS, we discovered statistically significant differences in the sulfation and acetylation patterns between populations. Since early diagnosis of RA is considered important in decelerating the disease's progression, identification of specific biomolecule characterizations may provide crucial information towards developing new therapies for suppressing the disease in its early stages. This is the first report of potential glycosaminoglycan biomarkers for RA found in human sera, while acknowledging the obvious fact that a larger population set, and more stringent collection parameters, will need to be investigated in the future.

Phosphorylation stoichiometries of human eukaryotic initiation factors.

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.

Differentiation of CC vs CXC chemokine dimers with GAG octasaccharide binding partners: an ion mobility mass spectrometry approach.

Chemokines, 8 kDa proteins implicated in leukocyte migration via oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation response as a means to regulate chemokine migration. Structural characterization of chemokines non-covalently bound to GAGs provides physiologically meaningful data in regard to routine inmmunosurveillance and disease response. In order to analyze the structures resulting from the GAG:chemokine interaction, we employed ion mobility mass spectrometry (IMMS) to analyze monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, and interleukin-8 (IL-8), a CXC chemokine, along with their individual interactions with GAG heparin octasaccharides. We show that MCP-1 and IL-8 are physiologically present as a dimer, with MCP-1 having two variants of its dimeric form and IL-8 having only one. We also show that the MCP-1 dimer adopts two conformations, one extended and one compact, when bound to a dodecasulfated heparin octasaccharide. Binding of MCP-1 to heparin octasaccharide isomers of varying sulfation patterns results in similar arrival time distribution values, which suggests minimal distinguishing features among the resultant complexes. Additionally, tandem mass spectrometry (MS/MS) showed that the binding of MCP-1 to a heparin octasaccharide has different dissociation patterns when compared with the corresponding IL-8 bound dimer. Overall, IMMS and MS/MS were used to better define the structural tendencies and differences associated with CC and CXC dimers when associated with GAG octasaccharides.

Novel mass spectrometric method for phosphorylation quantification using cerium oxide nanoparticles and tandem mass tags.

The stoichiometry of protein phosphorylation significantly impacts protein function. The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor the regulation levels of various proteins. This study reports an integrated methodology using cerium oxide nanoparticles and isobaric tandem mass tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation. This protocol was designed to directly measure the dephosphorylation levels for a known phosphorylation site, therefore allowing for quantification of phosphosites. Both the accuracy and precision of the method were verified using standard peptides and protein tryptic digests. This novel method was then applied to quantify phosphorylations on eukaryotic initiation factor 3H (eIF3H), a protein integral to overall eukaryotic protein translation initiation. To date, this is the first report of assessment of protein phosphorylation quantification on eIF3.

Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry.

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.

Phosphorylation of human eukaryotic initiation factor 2γ: novel site identification and targeted PKC involvement.

Eukaryotic translation requires a suite of proteins known as eukaryotic initiation factors (eIFs). These molecular effectors oversee the highly regulated initiation phase of translation. Essential to eukaryotic translation initiation is the protein eIF2, a heterotrimeric protein composed of the individually distinct subunits eIF2α, eIF2ß, and eIF2γ. The ternary complex, formed when eIF2 binds to GTP and Met-tRNA(i), is responsible for shuttling Met-tRNA(i) onto the awaiting 40S ribosome. As a necessary component for translation initiation, much attention has been given to the phosphorylation of eIF2α. Despite several previous investigations into eIF2 phosphorylation, most have centered on α- or ß-subunit phosphorylation and little is known regarding γ-subunit phosphorylation. Herein, we report eight sites of phosphorylation on the largest eIF2 subunit with seven novel phosphosite identifications via high resolution mass spectrometry. Of the eight sites identified, three are located in either the switch regions or nucleotide binding pocket domain. In addition, we have identified a possible kinase of eIF2, protein kinase C (PKC), which is capable of phosphorylating threonine 66 (thr-66) on the intact heterotrimer. These findings may shed new light on the regulation of ternary complex formation and alternate molecular effectors involved in this process prior to 80S ribosome formation and subsequent translation elongation and termination.

Distinct regions of human eIF3 are sufficient for binding to the HCV IRES and the 40S ribosomal subunit.

Translation of the hepatitis C virus (HCV) genomic RNA initiates from an internal ribosome entry site (IRES) in its 5' untranslated region and requires a minimal subset of translation initiation factors to occur, namely eukaryotic initiation factor (eIF) 2 and eIF3. Low-resolution structural information has revealed how the HCV IRES RNA binds human eIF3 and the 40S ribosomal subunit and positions the start codon for initiation. However, the exact nature of the interactions between the HCV IRES RNA and the translational machinery remains unknown. Using limited proteolysis and mass spectrometry, we show that distinct regions of human eIF3 are sufficient for binding to the HCV IRES RNA and the 40S subunit. Notably, the eIF3 subunit eIF3b is protected by HCV IRES RNA binding, yet is exposed in the complex when compared to subunits eIF3e, eIF3f, eIF3h, and eIF3l. Limited proteolysis reveals that eIF3 binding to the 40S ribosomal subunit occurs through many redundant interactions that can compensate for each other. These data suggest how the HCV IRES binds to specific regions of eIF3 to target the translational machinery to the viral genomic RNA and provide a framework for modeling the architecture of intact human eIF3.

Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.

The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins. Research presented herein is the first report showing retention of solution conformation of a protein as evidenced by CCS measurements of both recombinant and solvent disrupted versions of the same protein.

Identification of putative androgen receptor interaction protein modules: cytoskeleton and endosomes modulate androgen receptor signaling in prostate cancer cells.

We have developed a novel androgen receptor (AR) expression system in the 293 human embryonic kidney cell line that recapitulates AR biochemical activity as a steroid hormone receptor in prostate cancer cells. We used this system to identify putative AR-binding proteins in the cytosolic and nuclear compartments of mammalian cells using a large scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. For example, the heat shock 70 and 90 chaperones, which are known regulators of steroid hormone receptor, were identified as AR-binding proteins. AR purification enriched for proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link between AR and these protein modules in mammalian cells. For example, AR purification in the nuclear compartment led to the specific enrichment of alpha-actinin-4, clathrin heavy chain, and serine-threonine protein kinase C delta. Short interfering RNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, whereas alpha-actinin-4 and protein kinase C delta displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels. Lastly immunohistochemical staining of prostate tissue showed that alpha-actinin-4 levels decreased in the nucleus of high grade cancerous prostate samples, suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers. Taken together, these findings suggest AR binds to specific protein modules in mammalian cells and that these protein modules may provide a molecular framework for interrogating AR function in normal and cancerous prostate epithelial cells.

Evolution of 8p loss in transformed human prostate epithelial cells.

Deletion or rearrangement of sequences that map to the short arm of chromosome 8 (8p) are frequently associated with human prostate tumorigenesis. These losses often involve the entire short arm of chromosome 8 or very large regions of distal or proximal 8p, and several putative tumor suppressor genes mapping to 8p have been described. However, the mechanism responsible for 8p loss during prostate tumorigenesis has not been elucidated. In this study, we report data obtained using array comparative genomic hybridization and spectral karyotyping, which demonstrate successive translocation and deletion events responsible for loss of one copy of 8p in transformed human prostate epithelial cells. Moreover, this loss was accompanied by a pronounced transcriptional downregulation of genes mapping to the remaining copy of 8p and enhanced expression of traits associated with neoplastic transformation. Taken together, these studies illustrate a potential mechanism and functional role for 8p loss in human prostate tumorigenesis.

Molecular analysis of WFDC1/ps20 gene in prostate cancer.

BACKGROUND: WFDC1/ps20 protein has been previously established as a growth suppressor of the prostate cancer cell line PC3. It maps to chromosome 16q23.1, a region of frequent loss of heterozygosity, familial association, and genomic loss in prostate cancer. We, therefore, chose to examine WFDC1/ps20 for mutations and expression changes in prostate cancer. METHODS: DNA from 21 prostate cancer patients and 5 prostate cancer cell lines was screened for mutations in the WFDC1/ps20 gene by sequencing PCR products of each exon. An SphI polymorphism in the 5' UTR was screened in 23 tumors, 22 normal adjacent prostate tissue samples, and 35 control DNAs. Expression of WFDC1/ps20 in different tissue types was examined by Northern blot and by PCR across a multi-tissue cDNA panel. Expression patterns of WFDC1/ps20 in primary tumors were examined by full-length RT-PCR and products were cloned and sequenced to identify novel splice forms. Quantitative RT-PCR analysis of WFDC1/ps20 was performed in a separate cohort of matched tumor/benign tissues. RESULTS: No tumor-associated mutations were identified in the coding region of WFDC1/ps20. A novel polymorphism was found in exon 6 in DNA from cell lines, tumors, and normal adjacent benign tissue. A novel splice form completely deleted for exon 3 was found in tumor and normal prostate RNA. Quantitative RT-PCR demonstrated significant down regulation of WFDC1/ps20 in prostate tumors. Subdivision of normal tissue into stromal and epithelial compartments showed that WFDC1/ps20 expression correlates exponentially with the amount of stroma present. CONCLUSIONS: WFDC1/ps20 is down regulated but not frequently mutated in prostate cancer. It is expressed predominantly in the normal stroma of the prostate. We, therefore, propose that WFDC1/ps20 may not be a classical tumor suppressor gene, but might play a role in the maintenance of the normal extra cellular matrix milieu in the prostate.

Whole genome scanning identifies genotypes associated with recurrence and metastasis in prostate tumors.

Prostate cancer is the most commonly diagnosed non-cutaneous neoplasm among American males and is the second leading cause of cancer-related death. Prostate specific antigen screening has resulted in earlier disease detection, yet approximately 30% of men will die of metastatic disease. Slow disease progression, an aging population and associated morbidity and mortality underscore the need for improved disease classification and therapies. To address these issues, we analyzed a cohort of patients using array comparative genomic hybridization (aCGH). The cohort comprises 64 patients, half of whom recurred postoperatively. Analysis of the aCGH profiles revealed numerous recurrent genomic copy number aberrations. Specific loss at 8p23.2 was associated with advanced stage disease, and gain at 11q13.1 was found to be predictive of postoperative recurrence independent of stage and grade. Moreover, comparison with an independent set of metastases revealed approximately 40 candidate markers associated with metastatic potential. Copy number aberrations at these loci may define metastatic genotypes.

Integration of high-resolution array comparative genomic hybridization analysis of chromosome 16q with expression array data refines common regions of loss at 16q23-qter and identifies underlying candidate tumor suppressor genes in prostate cancer.

We have constructed a high-resolution genomic microarray of human chromosome 16q, and used it for comparative genomic hybridization analysis of 16 prostate tumors. We demarcated 10 regions of genomic loss between 16q23.1 and 16qter that occurred in five or more samples. Mining expression array data from four independent studies allowed us to identify 11 genes that were frequently underexpressed in prostate cancer and that co-localized with a region of genomic loss. Quantitative expression analyses of these genes in matched tumor and benign tissue from 13 patients showed that six of these 11 (WWOX, WFDC1, MAF, FOXF1, MVD and the predicted novel transcript Q9H0B8 (NM_031476)) had significant and consistent downregulation in the tumors relative to normal prostate tissue expression making them candidate tumor suppressor genes.

Evaluation of genetic patterns in different tumor areas of intermediate-grade prostatic adenocarcinomas by high-resolution genomic array analysis.

Prostate cancer is known for its highly heterogeneous histological appearance. Data concerning the cytogenetic content of areas with different histology are sparse. We have genetically evaluated 10 prostatic adenocarcinomas with intermediate histopathological grades (Gleason score 7) that showed two distinctive growth patterns with different pathologies, that is, Gleason grades 3 and 4 (G3 and G4). The G3 and G4 tumor specimens were taken from spatially separated regions within the cancer mass. Array-based comparative genomic hybridization (aCGH) was performed to obtain genotypes from the 10 pairs of G3 and G4 cancer areas. The cancer DNAs were retrieved from formalin-fixed and paraffin-embedded tissues allowing optimal recognition and selection of target cells. A genome-wide 2,400-element BAC array that provided high-resolution detection of both deletions and amplifications was used. In the 20 G3 and G4 areas, 252 genomic aberrations (88 gains, 164 deletions) were noted, of which 86 were concurrent in G3 and G4 areas (34% overlap). Ninety-five of the 252 alterations were defined by a single BAC clone (54 gains, 41 deletions). Overlapping changes were more frequent for deletions (46%) than for gains (13%). Frequent coinciding deletions (> or = 20% of tumors) were seen on 8p (60%), 6q (30%), 1p (20%), 2q (20%), proximal 8q (20%), 10q (20%), 13q (20%), 16q (20%), and 18q (20%). A frequent overlapping gain (> or = 20% of tumors) was detected on distal 13q (20%). The patterns of imbalance could be found to coincide in the G3 and G4 areas of the majority of cancers. Array-based CGH can be used as a tool for the evaluation of genetic patterns in prostate cancer.

High-resolution analysis of paraffin-embedded and formalin-fixed prostate tumors using comparative genomic hybridization to genomic microarrays.

We have used prostate cancer, the most commonly diagnosed noncutaneous neoplasm among men, to investigate the feasibility of performing genomic array analyses of archival tissue. Prostate-specific antigen and a biopsy Gleason grade have not proven to be accurate in predicting clinical outcome, yet they remain the only accepted biomarkers for prostate cancer. It is likely that distinct spectra of genomic alterations underlie these phenotypic differences, and that once identified, may be used to differentiate between indolent and aggressive tumors. Array comparative genomic hybridization allows quantitative detection and mapping of copy number aberrations in tumors and subsequent associations to be made with clinical outcome. Archived tissues are needed to have patients with sufficient clinical follow-up. In this report, 20 formalin-fixed and paraffin-embedded prostate cancer samples originating from 1986 to 1996 were studied. We present a straightforward protocol and demonstrate the utility of archived tissue for array comparative genomic hybridization with a 2400 element BAC array that provides high-resolution detection of both deletions and amplifications.