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.
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.
Acylnitroso cycloadducts have proven to be valuable intermediates in the syntheses of a plethora of biologically active molecules. Recently, organometallic reagents were shown to open bicyclic acylnitroso cycloadducts and, more interestingly, the prospect of highly regioselective openings was raised. This transformation was employed in the synthesis of a compound with excellent inhibitory activity against 5-lipoxygenase ((±)-4a, IC50 51 nM), an important mediator of inflammation intimately involved in a number of disease states including asthma and cancer. Optimization of the copper-mediated organometallic ring opening reaction was accomplished allowing the further exploration of the biological activity. Synthesis of a number of derivatives with varying affinity for metal binding as well as pendant groups in a range of sizes was accomplished. Analogues were tested in a whole cell assay which revealed a subset of the compounds to be inhibitors of enzyme translocation, a mode of action not previously known and, potentially, extremely important for better understanding of the enzyme and inhibitor development. Additionally, the lead compound was tested in vivo in an established colon cancer model and showed very encouraging anti-tumorogenic properties.
Regioselective ring opening of N-hydroxycarbamate-derived nitroso cycloadducts by a coppercatalyzed allylic alkylation reaction was achieved and applied to the synthesis of a set of substituted diaryl ether containing compounds. Use of protected 3-hydroxybenzyl bromide allowed access to a late stage phenol intermediate after protection of the N-hydroxy moiety that was generated from the ring opening reaction. The diaryl ethers were then formed by coppermediated coupling with arylboronic acids. After selective deprotection, alumina-promoted transcarbamoylation provided the target compounds. Previous results indicate the compounds may possess significant inhibitory potency against the proinflammatory enzyme 5-lipoxygenase. KeywordsRegioselective Ring-opening; HeteroDiels-Alder; Bisaryl ether; transcarbamoylation; lipoxygenase Hydroxamic acids, hydroxamates, N-hydroxycarbamates, and N-hydroxyureas are important pharmacophores due to their ability to bind biologically relevant metals such as iron and zinc. 1 Previously our lab described Grignard-mediated ring opening reactions of Nhydroxycarbamate-derived nitroso cycloadducts that led to the synthesis of an Nhydroxycarbamate (ND-6008) with potent inhibitory activity against the iron-containing enzyme 5-lipoxygenase. 2 5-Lipoxygenase, an important mediator of inflammation, 3 has been the focus of numerous drug discovery programs for the treatment of diseases ranging from asthma to cancer. 4 We sought to expand upon this result through the development of a divergent synthetic route that would allow the facile syntheses of structurally and electronically diverse analogs. We recently reported synthetic variations of the metal binding groups and found that representative compounds potently inhibit 5-lipoxygenase translocation. 5 Herein, report on synthetic elaboration of analogs containing substituents on the distal ring of the diaryl ether.Initial studies by our group demonstrated that, while acylnitroso and N-hydroxycarbamatederived nitroso cycloadducts could be opened by Grignard reagents, the presence of catalytic copper was essential for high conversion. 6 These results were later expanded upon and the * Corresponding author. Tel.: 574-631-7571; fax: 574-631-6652;, mmiller1@nd.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The CuCN-catalyzed reaction of methyl and t-butyl N-hydroxycarbamate derived cycloadducts 1 with benzyl magnesium chloride was studied with respect to catalyst loading, addition time of the Grignard reagent and temperature (Table 1). When the Grignard reagent was added to the cycloadduct and...
Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351
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