Karl A Walter

Publications (12)45.83 Total impact

• Article: Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design.

  Enrico L Digiammarino, John E Harlan, Karl A Walter, Uri S Ladror, Rohinton P Edalji, Charles W Hutchins, Marc R Lake, Amy J Greischar, Junjian Liu, Tariq Ghayur, Clarissa G Jakob
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  ABSTRACT: The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.
  mAbs 09/2011; 3(5):487-94.
• Source

  Available from: Alla Korepanova

  Article: Expression and purification of human TRPV1 in baculovirus-infected insect cells for structural studies.

  Alla Korepanova, Ana Pereda-Lopez, Larry R Solomon, Karl A Walter, Marc R Lake, Bruce R Bianchi, Heath A McDonald, Torben R Neelands, Jianwei Shen, Edmund D Matayoshi, Robert B Moreland, Mark L Chiu
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  ABSTRACT: TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.
  Protein Expression and Purification 01/2009; 65(1):38-50. · 1.59 Impact Factor
• Article: Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance.

  Jeffrey R Huth, Danying Song, Renaldo R Mendoza, Candice L Black-Schaefer, Jamey C Mack, Sarah A Dorwin, Uri S Ladror, Jean M Severin, Karl A Walter, Diane M Bartley, Philip J Hajduk
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  ABSTRACT: We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.
  Chemical Research in Toxicology 01/2008; 20(12):1752-9. · 3.78 Impact Factor
• Article: Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies.

  Jeffrey R Huth, Chang Park, Andrew M Petros, Aaron R Kunzer, Michael D Wendt, Xilu Wang, Christopher L Lynch, Jamey C Mack, Kerry M Swift, Russell A Judge, [......], Stephen K Tahir, Edward D Matayoshi, Sarah A Dorwin, Uri S Ladror, Jean M Severin, Karl A Walter, Diane M Bartley, Stephen W Fesik, Steven W Elmore, Philip J Hajduk
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  ABSTRACT: The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.
  Chemical Biology &amp Drug Design 08/2007; 70(1):1-12. · 2.28 Impact Factor
• Article: From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

  Claude G Lerner, Philip J Hajduk, Rolf Wagner, Frank L Wagenaar, Charlotte Woodall, Yu-Gui Gu, Xenia B Searle, Alan S Florjancic, Tianyuan Zhang, Richard F Clark, [......], Annapur G Shivakumar, Anne Y Saiki, Niru Soni, Mai Bui, Darlene J Balli, William J Sanders, Angela M Nilius, Thomas F Holzman, Stephen W Fesik, Bruce A Beutel
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  ABSTRACT: As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.
  Chemical Biology &amp Drug Design 07/2007; 69(6):395-404. · 2.28 Impact Factor
• Article: Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-RS.

  Chaohong Sun, Danying Song, Rachel A Davis-Taber, Leo W Barrett, Victoria E Scott, Paul L Richardson, Ana Pereda-Lopez, Marie E Uchic, Larry R Solomon, Marc R Lake, Karl A Walter, Philip J Hajduk, Edward T Olejniczak
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  ABSTRACT: The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-R(S)) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-R(S) with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed beta-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-R(S). These results present a structural basis for hPAC1-R(S) selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-R(S) receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.
  Proceedings of the National Academy of Sciences 06/2007; 104(19):7875-80. · 9.68 Impact Factor
• Article: Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

  Wende Niforatos, Xu-Feng Zhang, Marc R Lake, Karl A Walter, Torben Neelands, Thomas F Holzman, Victoria E Scott, Connie R Faltynek, Robert B Moreland, Jun Chen
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  ABSTRACT: As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.
  Molecular Pharmacology 06/2007; 71(5):1209-16. · 4.88 Impact Factor
• Article: Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

  Jun Chen, Marc R Lake, Reza S Sabet, Wende Niforatos, Steve D Pratt, Steven C Cassar, Jing Xu, Sujatha Gopalakrishnan, Ana Pereda-Lopez, Murali Gopalakrishnan, Thomas F Holzman, Robert B Moreland, Karl A Walter, Connie R Faltynek, Usha Warrior, Victoria E Scott
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  ABSTRACT: Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.
  Journal of Biomolecular Screening 03/2007; 12(1):61-9. · 2.05 Impact Factor
• Article: Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure.

  Kenton L Longenecker, Geoffrey F Stamper, Philip J Hajduk, Elizabeth H Fry, Clarissa G Jakob, John E Harlan, Rohinton Edalji, Diane M Bartley, Karl A Walter, Larry R Solomon, Thomas F Holzman, Yu Gui Gu, Claude G Lerner, Bruce A Beutel, Vincent S Stoll
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  ABSTRACT: In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.
  Protein Science 01/2006; 14(12):3039-47. · 2.80 Impact Factor
• Source

  Available from: harvardapparatus.com

  Article: Kringle 5 of human plasminogen induces apoptosis of endothelial and tumor cells through surface-expressed glucose-regulated protein 78.

  Don J Davidson, Catherine Haskell, Sandy Majest, Abdullah Kherzai, David A Egan, Karl A Walter, Andrew Schneider, Earl F Gubbins, Larry Solomon, Zhebo Chen, Rick Lesniewski, Jack Henkin
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  ABSTRACT: Kringle 5 (K5) of human plasminogen has been shown to inhibit angiogenesis by inducing the apoptosis of proliferating endothelial cells. Peptide regions around the lysine-binding pocket of K5 largely mediate these effects, particularly the peptide PRKLYDY, which we show to compete with K5 for the binding to endothelial cells. The cell surface binding site for K5 that mediates these effects has not been defined previously. Here, we report that glucose-regulated protein 78, exposed on cell surfaces of proliferating endothelial cells as well as on stressed tumor cells, plays a key role in the antiangiogenic and antitumor activity of K5. We also report that recombinant K5-induced apoptosis of stressed HT1080 fibrosarcoma cells involves enhanced activity of caspase-7, consistent with the disruption of glucose-regulated protein 78-procaspase-7 complexes. These results establish recombinant K5 as an inhibitor of a stress response pathway, which leads to both endothelial and tumor cell apoptosis.
  Cancer Research 07/2005; 65(11):4663-72. · 7.86 Impact Factor
• Article: Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

  William J Sanders, Vicki L Nienaber, Claude G Lerner, J Owen McCall, Sean M Merrick, Susan J Swanson, John E Harlan, Vincent S Stoll, Geoffrey F Stamper, Stephen F Betz, Kevin R Condroski, Robert P Meadows, Jean M Severin, Karl A Walter, Peter Magdalinos, Clarissa G Jakob, Rolf Wagner, Bruce A Beutel
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  ABSTRACT: Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.
  Journal of Medicinal Chemistry 04/2004; 47(7):1709-18. · 5.25 Impact Factor
• Article: Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery.

  Kent D Stewart, Kevin Steffy, Kevin Harris, John E Harlan, Vincent S Stoll, Jeffrey R Huth, Karl A Walter, Emily Gramling-Evans, Renaldo R Mendoza, Jean M Severin, Paul L Richardson, Leo W Barrett, Edmund D Matayoshi, Kerry M Swift, Stephen F Betz, Steve W Muchmore, Dale J Kempf, Akhter Molla
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  ABSTRACT: Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.
  Journal of Computer-Aided Molecular Design 21(1-3):121-30. · 3.39 Impact Factor