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1.  Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors 
Bioorganic & medicinal chemistry  2013;22(1):616-622.
In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics.
doi:10.1016/j.bmc.2013.10.039
PMCID: PMC3917480  PMID: 24286762
2.  Calpain 10 Homology Modeling with CYGAK and Increased Lipophilicity Leads to Greater Potency and Efficacy in Cells 
ACS chemical biology  2012;7(8):1410-1419.
Calpain 10 is a ubiquitously expressed mitochondrial and cytosolic Ca2+-regulated cysteine protease in which overexpression or knockdown leads to mitochondrial dysfunction and cell death. We previously identified a potent and specific calpain 10 peptide inhibitor (CYGAK), but it was not efficacious in cells. Therefore, we created a homology model using the calpain 10 amino acid sequence and calpain 1 3-D structure and docked CYGAK in the active site. Using this model we modified the inhibitor to improve potency 2-fold (CYGAbuK). To increase cellular efficacy, we created CYGAK-S-phenyl-oleic acid heterodimers. Using renal mitochondrial matrix CYGAK, CYGAK-OC, and CYGAK-ON had IC50’s of 70, 90, and 875 nM, respectively. Using isolated whole renal mitochondria CYGAK, CYGAK-OC, and CYGAK-ON had IC50’s of 95, 196, and >10,000 nM, respectively. Using renal proximal tubular cells (RPTC) in primary culture, 30 min exposures to CYGAK-OC and CYGAbuK-OC decreased cellular calpain activity approximately 20% at 1 μM, and concentrations up to 100 μM had no additional effect. RPTC treated with 10 μM CYGAK-OC for 24 h induced accumulation of ATP synthase β and NDUFB8, two calpain 10 substrates. In summary, we used molecular modeling to improve the potency of CYGAK, while creating CYGAK-oleic acid heterodimers to improve efficacy in cells. Since calpain 10 has been implicated in type 2 diabetes and renal aging, the use of this inhibitor may contribute to elucidating the role of calpain 10 in these and other diseases.
doi:10.1021/cb300219h
PMCID: PMC4251666  PMID: 22612451
3.  Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly 
Journal of medicinal chemistry  2013;56(4):1573-1582.
A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds.
doi:10.1021/jm3013882
PMCID: PMC3692612  PMID: 23323521
4.  Structural Characterization of the Binding Interactions of Various Endogenous Estrogen Metabolites with Human Estrogen Receptor α and β Subtypes: A Molecular Modeling Study 
PLoS ONE  2013;8(9):e74615.
In the present study, we used the molecular docking approach to study the binding interactions of various derivatives of 17β-estradiol (E2) with human estrogen receptor (ER) α and β. First, we determined the suitability of the molecular docking method to correctly predict the binding modes and interactions of two representative agonists (E2 and diethylstilbesterol) in the ligand binding domain (LBD) of human ERα. We showed that the docked structures of E2 and diethylstilbesterol in the ERα LBD were almost exactly the same as the known crystal structures of ERα in complex with these two estrogens. Using the same docking approach, we then characterized the binding interactions of 27 structurally similar E2 derivatives with the LBDs of human ERα and ERβ. While the binding modes of these E2 derivatives are very similar to that of E2, there are distinct subtle differences, and these small differences contribute importantly to their differential binding affinities for ERs. In the case of A-ring estrogen derivatives, there is a strong inverse relationship between the length of the hydrogen bonds formed with ERs and their binding affinity. We found that a better correlation between the computed binding energy values and the experimentally determined logRBA values could be achieved for various A-ring derivatives by re-adjusting the relative weights of the van der Waals interaction energy and the Coulomb interaction energy in computing the overall binding energy values.
doi:10.1371/journal.pone.0074615
PMCID: PMC3786999  PMID: 24098659
5.  Targeting sub-cellular localization through the Polo-Box Domain: non-ATP competitive Inhibitors recapitulate a PLK1 phenotype 
Molecular cancer therapeutics  2012;11(8):1683-1692.
The polo-box domain (PBD) has critical roles in the mitotic functions of PLK1. The REPLACE strategy to develop inhibitors of protein-protein interactions has identified alternatives for the N-terminal tripeptide of a Cdc25C substrate. In addition, a peptide structure activity relationship described key determinants and novel information useful for drug design. Fragment ligated inhibitory peptides (FLIPs) were generated with comparable affinity to peptide PBD inhibitors and possessed anti-proliferative phenotypes in cells consistent with the observed decrease in PLK1 centrosomal localization. These FLIPs demonstrated evidence of enhanced PLK1 inhibition in cells relative to peptides and induced monopolar and multipolar spindles, which stands in contrast to previously reported small molecule PBD inhibitors that display phenotypes only partially representative of PLK1 knockdown. Progress obtained applying REPLACE validates this approach for identifying fragment alternatives for determinants of the Cdc25C binding motif and extends its applicability of the strategy for discovering protein-protein interaction inhibitors. In addition, the described PBD inhibitors retain high specificity for PLK1 over PLK3 and therefore show promise as isotype selective, non-ATP competitive kinase inhibitors that provide new impetus for the development of PLK1 selective anti-tumor therapeutics.
doi:10.1158/1535-7163.MCT-12-0006-T
PMCID: PMC3711794  PMID: 22848093
Phosphorylation and proteolysis in cell cycle control; Protein serine-threonine kinases; Molecular modelling; In silico evaluation of targets and design of libraries; Protein/protein interactions; Cell cycle mechanisms of anticancer drug action; Kinase and phosphatase inhibitors; Novel antitumor agents
6.  Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements 
ACS chemical biology  2010;5(12):1169-1182.
An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove which to date has not been carried out in a systematic fashion. Collectively, the data presented provides new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and anti-tumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.
doi:10.1021/cb1001262
PMCID: PMC3425359  PMID: 20843055
7.  Functional characterization and identification of mouse Rad51d splice variants 
BMC Molecular Biology  2009;10:27.
Background
The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns.
Results
Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DΔ7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dΔ3 (deleted for exon 3) and Rad51dΔ5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dΔ3 being detected in each tissue except ovary and Rad51dΔ5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.
Conclusion
These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.
doi:10.1186/1471-2199-10-27
PMCID: PMC2667185  PMID: 19327148

Results 1-7 (7)