Abstracts D1 - D5

Category D.  Protein Structure & Function

 

D1.  Pursuing Lead Compounds to Disrupt the MEF2:HDAC4 Protein Complex: A Potential Route for Huntington’s Disease Therapeutics

Nurjahan Mehzabeen1, Anne Cooper1, Na Zhang1, Fei Philip Gao1, Kevin P. Battaile2, Anna Rzepiela4, Vahri Beaumont3, Ignacio Muñoz-Sanjuán3, Celia Dominguez3, Alex Kiselyov3, Michel Maillard3 and Scott Lovell1

1Del Shankel Structural Biology Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047
2IMCA-CAT, APS, Argonne National Laboratory Argonne, IL 60439
3CHDI Foundation Inc., Los Angeles, CA 90045.
4Pyxis, Delft, The Netherlands

Recent studies have shown that transcriptional dysregulation is a major process involved in the pathology of Huntington’s disease, an inherited neurodegenerative disorder characterized by the presence of a mutant form of the huntingtin protein. It has been suggested that the mutant huntingtin protein affects histone acetyltransferase activity resulting in a reduction in histone acetylation causing repressed transcription of particular genes necessary for neuronal cell survival in HD patients. One approach to combat this transcriptional dysregulation is to inhibit the catalytic activity of histone deacetylases, such as HDAC4, thereby allowing unregulated expression of certain genes that promote neuronal cell survival in Huntington’s disease patients. However, the effectiveness of current inhibitors of HDAC4 is limited due to their toxicity.

A potential alternative approach is to inhibit the interaction between HDAC4 and respective protein partners such as Myocyte Enhancer Factor 2 (MEF2) as its transcription factor activity is negatively regulated by HDAC4. In an effort to identify lead candidate compounds that target the HDAC4:MEF2 complex, select compounds that could potentially occupy the HDAC4 binding pocket of MEF2, were identified using in silico screening.  Surface plasmon resonance (SPR) experiments were conducted to screen the pool of compounds against two isoforms of MEF2 (MEF2A and MEF2D) and structural characterization of the binding modes for the top hits was attempted.  However, electron density consistent with the compounds was not observed for samples prepared by cocrystallization or soaking.  Analysis of the crystal packing of the MEF2D structure revealed that an alpha helix from a symmetry related molecule occupies the HDAC4 binding pocket and may prevent structural characterization of the ligand binding mode.  Interestingly, this alpha helix adopts a similar binding orientation as HDAC9, observed in a previously characterized HDAC9:MEF2 structure.  Three top compound hits were identified that will need to undergo further validation prior to their development into lead inhibitors of MEF2.

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D2.  PvdF as potential novel transformylase from Pseudomonas aeruginosa

Nikola Kenjic and Audrey L. Lamb

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA

Siderophores are bioactive peptides responsible for iron scavenging by bacteria and fungi in iron limiting environments. In opportunistic pathogens like Pseudomonas aeruginosa, pyoverdin is just one of the siderophores made. The enzymatic biosynthesis of these molecules is characterized by “assembly line” production by nonribosomal peptide synthetases (NRPS). One of the amino acids incorporated into pyoverdin is an ornithine that has been modified by the PvdA, a documented ornithine hydroxylase, and PvdF enzymes. Based on the gene context, it has been proposed that PvdF catalyzes the conversion of hydroxyornithine to formyl - hydroxyornithine using N10-formyltetrahydrofolate (N10-THF) as the formyl donor. Here we demonstrate that a more stable analogue of N10-THF fDDf (5,8 dideazafolate ) can be used to initiate enzymatic turnover. We also have preliminary crystals of PvdF that diffract to ~4Å. This preliminary evidence will serve as the basis for my dissertation project, determining the structure function relationship for this yet uncharacterized class of enzymes.

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D3.  Structure-based Discovery and Functional Characterization of a Type III Secretion Regulator in Chlamydia trachomatis

Michael L. Barta1Jason R. Wickstrum1, Robert J. Suchland2, Frances S. Mandelbaum1, P. Scott Hefty*1

1Department of Molecular Biosciences, University of Kansas,
2Department of Internal Medicine, University of Washington, Seattle, Washington

Background: A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins). The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function within the medically important, obligate intracellular human pathogen Chlamydia trachomatis.

Methods/Results: A crystal structure of CT398 was determined that displayed a high degree of structural similarity to FlgZ (Flagellar-associated zinc-ribbon domain protein) from Helicobacter pylori. This observation directed analyses of candidate protein partners of CT398, revealing interactions with two paralogous Type III Secretion System (T3SS) ATPase regulators (CdsL and FliH) and the alternative sigma factor RpoN (σ54). The functional role of CT398 was further examined by conditional gene expression and subsequent phenotypic evaluation.  Overexpression of CT398 during the developmental cycle resulted in significantly decreased inclusion volume, reduced levels of infectious progeny and grossly enlarged morphology. Furthermore, this aberrant state was correlated with a temporal deregulation of T3S effector protein translocation directly caused by increased expression of CT398.

Conclusions: These studies provide the first phenotypic analyses of overexpressed genes in Chlamydia, and suggest that CT398 functions in several key areas of chlamydial pathogenesis, including in the regulation of T3SS activity, potentially mediated by CT398-mRNA interactions at the T3S export apparatus. We thus propose that CT398 be named CdsZ (Contact-dependent secretion-associated zinc-ribbon domain protein).

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D4.  Structure-metabolism relationships of novel macrocyclic peptide opioid receptor ligands

T. Khaliq1, S. N. Senadheera1, S. M. Lunte2 and J. V. Aldrich1

1Department of Medicinal Chemistry,  2Department of Pharmaceutical Chemistry, the University of Kansas, Lawrence, KS, 66045 USA

We are pursuing metabolically stable peptidic ligands for kappa opioid receptors (KOR) as potential treatments for drug abuse and pain. We synthesized the natural product macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]), which was reported to be a KOR antagonist in vitro,1 and found that it displays mixed agonist/KOR antagonism in vivo.2 Both CJ-15,208 and its D-Trp isomer are active after oral administration and appear to penetrate into the CNS3,4,5 making these peptides promising agents for further development. In order to optimize their structures, we have examined the metabolic stabilities of the lead peptides in blood and in liver microsomes. Both peptides were completely stable in blood for at least 20 h. CJ-15,208 was substantially more stable in liver microsomes than its D-Trp isomer. The evaluation of metabolic stabilities in liver microsomes of a series of both L-Trp and D-Trp analogs revealed that the stereochemistry of the tryptophan residue is critical in determining the metabolic stability of these macrocyclic tetrapeptides. The results from these metabolism studies are guiding the design of additional analogs with improved pharmacokinetic properties. This research was supported by NIDA grants R01 DA023924 and R01 DA032928.

1. T. Saito et al., J. Antibiot. 2002, 55, 847.
2. N. C. Ross et al., Br. J. Pharmacol. 2012, 165, 1097.
3. S. N. Senadheera et al., In Peptides: Building Bridges, Lebl, M., Ed. American Peptide Society: San Diego, CA, 2011, 346.
4. S. O. Eans et al., Br. J. Pharmacol. 2013, 169, 426.
5. J. V. Aldrich et al., J. Nat. Prod. 2013, 76, 433.

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D5.  The University of Kansas Protein Structure Laboratory

Nurjahan Mehzabeen and Scott Lovell

Protein Structure Core Laboratory, Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF), University of Kansas, Lawrence, Kansas
Del Shankel Structural Biology Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047

The Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF) at The University of Kansas University supports health related basic research efforts to obtain structural/functional information for proteins. One of the three core laboratories at the COBRE-PSF, the Protein Structure Laboratory (PSL), collaborates with investigators from various institutions in an effort to obtain the 3-dimensional structures of proteins using X-ray crystallography. The capabilities and infrastructure of the PSL are presented here along with examples of collaborative projects that have been completed. The results from these projects highlight the importance of obtaining structural information to provide mechanistic/functional insight for particular proteins and demonstrate the significance of structural biology to facilitate and support drug discovery efforts.

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