Kirk Clark

Characterization of human PLD2 and the analysis of PLD isoform splice variants

Phospholipase D (PLD) cleaves phosphatidylcholine in response to a variety of cell stimuli to release phosphatidic acid, which is associated with a number of cellular responses including regulated secretion, mitogenesis, and cytoskeletal changes. Recent advances in this field include the reports of cDNA sequences for two mammalian PLD isoforms: human PLD1 and rodent PLD2. We report the characterization of cDNA encoding human PLD2. In these experiments, we uncovered alternate splice variants of both human isoforms and evaluated the relative abundance of these messages by reverse transcriptase polymerase chain reaction, thereby indicating the physiologically relevant forms. Further, Northern hybridization experiments defined the tissue distribution of the human PLD messages. Human PLD1 does not appear to be an abundant message in any tissue tested whereas levels of human PLD2 mRNA apparently were higher and more variable. The specific activity and regulation of recombinant human PLD2 are indistinguishable from that of recombinant mouse PLD2. Analysis of the amino acid sequences of both human isoforms revealed important putative Pleckstrin homology domains and identified additional members of the PLD gene family that help to delimit the catalytic domain. The presence of Pleckstrin homology domains in the PLDs resolves several contradictory observations regarding PLD regulation and the domain structure of the proteins.— Steed, P. M., Clark, K. L., Boyar, W. C., Lasala, D. J. Characterization of human PLD2 and the analysis of PLD isoform splice variants. FASEB J. 12, 1309–1317 (1998)

N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors with Antibacterial Activity

ABSTRACT Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P1′ site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P1′ site. Compounds with MICs of ≤4 μg/ml against gram-positive and gram-negative pathogens, including Staphylococcusaureus, Streptococcuspneumoniae, and Haemophilusinfluenzae, have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC50s) for Escherichiacoli Ni-PDF were ≤0.1 μM, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC50s of consistently >200 μM for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 Å. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

Histone-like transcription factors in eukaryotes.

Histone proteins have long been recognized as important regulators of eukaryotic gene expression. Condensation of DNA into chromatin by the core (H2A, H2B, H3, H4) and linker (H1, H5) histones effectively represses transcription initiation from the promoters of genes that have been packaged. Recently, eukaryotic transcriptional activators and coactivators (both positive and negative) resembling core and linker histone proteins have been discovered. Substantial progress has been made on structural and mechanistic studies of histones and histone-like transcription factors. Three-dimensional structures solved include the core histone octamer, an archael histone homodimer, two core histone-like subunits of transcription factor IID, a linker histone, and a linker histone-like transcriptional activator.

Synthesis of a well-defined glycoconjugate vaccine by a tyrosine-selective conjugation strategy

An anti-candidiasis glycoconjugate vaccine was prepared via a tyrosine-selective alkynylation and a click chemistry mediated glycoconjugation sequence. It features a well-defined glycan, protein carrier, and connectivity. The construct, although with significantly lower carbohydrate loading and a shorter β-(1,3) glucan chain than the well-established anti-candidiasis vaccine derived from the random conjugation of laminarin at lysines, elicited a comparable level of specific IgG antibodies.

Peptide deformylase inhibitors of Mycobacterium tuberculosis: synthesis, structural investigations, and biological results.

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.

Potent and selective 2-naphthylsulfonamide substituted hydroxamic acid inhibitors of matrix metalloproteinase-13.

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.

Abstract 3853: TAS266, a novel tetrameric nanobody agonist targeting death receptor 5 (DR5), elicits superior antitumor efficacy than conventional DR5-targeted approaches

Preferential induction of apoptosis in cancer cells has been the objective of therapeutic strategies targeting apoptotic pathways. To this end, multiple therapeutic agonists of Death Receptors 4 and 5 (DR4, DR5), have been developed and are under clinical evaluation. Although these agonists, including antibodies and soluble ligand TRAIL, demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation for the discrepant pre-clinical and clinical results is that DR5 may play a more prominent role in in vitro model systems as opposed to cancers in humans. Alternatively, these results might indicate that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients. In particular, naturally dimeric antibody agonists require secondary cross-linking via Fcα receptors expressed on immune cells present in the tumor microenvironment to achieve optimal clustering of DR5 into a ternary active state. Because immune cell content in the tumor can be heterogeneous, reliance on this secondary mechanism for activity may limit the potency of these antibodies. To overcome this limitation, a novel nanobody approach was taken to eliminate the need for cross-linking and improve receptor activation with the goal of generating a significantly more potent DR5 agonist. Nanobodies are a class of therapeutic proteins based on single, high affinity heavy chain domain (VHH) antibodies that naturally occur in camelid species, and these VHH domains can be linked to form multivalent structures (di-, tri-, tetra-, etc). This approach led to the development of a tetrameric DR5 targeted agonist, TAS266, with significantly greater avidity for DR5 binding. TAS266 activates downstream caspases with more rapid kinetics and is up to 1000-fold more potent in cell death assays when compared to a cross-linked DR5 antibody or TRAIL. In vivo, TAS266 elicits single dose tumor regressions in multiple tumor xenograft models and sustained tumor regressions after treatment cessation. TAS266 showed superior anti-tumor activity compared to a DR5 agonist antibody and TRAIL, including the ability to induce tumor regression in a patient-derived primary pancreatic tumor model that is insensitive to the agonist antibody. Thus, TAS266 has the potential for superior clinical activity in settings insensitive to the conventional therapeutic approaches to DR5. First-in-man trials are expected to begin in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3853. doi:1538-7445.AM2012-3853

Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Full-length myocilin protein is purified from mammalian cells as a dimer

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.