Name: development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors
Uploaded: 2015-10-26T14:00:00
Description: Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

We thank Dr&#8217;s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.

1. Computational Identification of Potential Small Molecule Capping Groups
Note: In principle, a variety of docking or pharmacophore search methods can be used to predict potential capping groups. The main purpose of computational studies in REPLACE is to identify small molecules that retain the features and interactions of the amino acids that are substituted.
<ol>
	<li>Validation of the LigandFit docking protocol14</li>
</ol>
Note: In previous studies, th...

<ol>
	<li>Andrews, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REPLACE:+a+strategy+for+iterative+design+of+cyclin-binding+groove+inhibitors.">REPLACE: a strategy for iterative design of cyclin-binding groove inhibitors.</a> Chembiochem. 7, 1909-1915 (2006).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Non-ATP+Competitive+CDK/Cyclin+Groove+Inhibitors+through+REPLACE-Mediated+Fragment+Assembly.">Optimization of Non-ATP Competitive CDK/Cyclin Groove Inhibitors through REPLACE-Mediated Fragment Assembly.</a> J Med Chem. 56, 1573-1582 (2013).</li><li>McInnes, C., Bernstein, M., Desai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+29.">Chapter 29.</a> Annual Reports in Medicinal Chemistry. 47, 459-474 (2012).</li><li>Bower, J. F., Pannifer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fragment-based+technologies+to+target+protein-protein+interactions.">Using fragment-based technologies to target protein-protein interactions.</a> Curr Pharm Des. 18, 4685-4696 (2012).</li><li>Makley, L. N., Gestwicki, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+number+of+'druggable'+targets:+non-enzymes+and+protein-protein+interactions.">Expanding the number of 'druggable' targets: non-enzymes and protein-protein interactions.</a> Chemical biology, and drug design. 81, 22-32 (2013).</li><li>Walensky, L. D., Bird, G. 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J., Zheleva, D. I., Lane, D. P., Fischer, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomimetic+design+of+CDK+inhibitors+targeting+the+recruitment+site+of+the+cyclin+subunit.">Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.</a> Current medicinal chemistry. Anti-cancer agents. 3, 57-69 (2003).</li><li>Premnath, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragment+based+discovery+of+arginine+isosteres+through+REPLACE:+towards+non-ATP+competitive+CDK+inhibitors.">Fragment based discovery of arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors.</a> Bioorganic, and medicinal. 22, 616-622 (2014).</li><li>Venkatachalam, C. M., Jiang, X., Oldfield, T., Waldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LigandFit:+a+novel+method+for+the+shape-directed+rapid+docking+of+ligands+to+protein+active+sites.">LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites.</a> J Mol Graph Model. 21, 289-307 (2003).</li><li>Mendoza, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cyclin-dependent+kinase+2/cyclin+A+antagonists+that+differ+from+ATP+site+inhibitors+block+tumor+growth.">Selective cyclin-dependent kinase 2/cyclin A antagonists that differ from ATP site inhibitors block tumor growth.</a> Cancer Res. 63, 1020-1024 (2003).</li><li>Denizot, F., Lang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cell+growth+and+survival.+Modifications+to+the+tetrazolium+dye+procedure+giving+improved+sensitivity+and+reliability.">Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.</a> J Immunol Methods. 89, 271-277 (1986).</li><li>Zheleva, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+potent+p21(WAF1)-derived+peptide+inhibitors+of+CDK-mediated+pRb+phosphorylation:+delineation+and+structural+insight+into+their+interactions+with+cyclin+A.">Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.</a> The journal of peptide research : official journal of the American Peptide Society. 60, 257-270 (2002).</li><li>Ivanov, A. A., Khuri, K. F. R., Fu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein-protein+interactions+as+an+anticancer+strategy.">Targeting protein-protein interactions as an anticancer strategy.</a> Trends in Pharmacological Sciences. 34, (2013).</li><li>Thayer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+peptides.">Improving peptides.</a> Chemical and Engineering News. 89, 13-20 (2011).</li><li>Yin, H., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+targeting+protein-protein+interactions+with+synthetic+agents.">Strategies for targeting protein-protein interactions with synthetic agents.</a> Angew Chem Int Ed Engl. 44, 4130-4163 (2005).</li><li>Jubb, H., Higueruelo, A. P., Winter, A., Blundell, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+and+drug+discovery+for+protein-protein+interactions.">Structural biology and drug discovery for protein-protein interactions.</a> Trends Pharmacol Sci. 33, 241-248 (2012).</li><li>Cannon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceutics+and+drug+delivery+aspects+of+heme+and+porphyrin+therapy.">Pharmaceutics and drug delivery aspects of heme and porphyrin therapy.</a> J Pharm Sci. 82, 435-446 (1993).</li></ol>

Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI&#8217;s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and...

In this article, a case study of applying the REPLACE (Replacement with partial ligand alternatives using computational enrichment) strategy to convert peptidic inhibitors of protein-protein interactions into more pharmaceutically relevant molecules is described1-3. While PPIs represent a rich but underexploited source of potential drug targets, existing methodologies are largely insufficient to make these widely accessible. Current strategies including fragment based design4, high-throughput screening5 and stapled peptides6 have provided advances, however these are in many cases ineffective. As a result, more progress and more efficient approaches are required. REPLACE has been fully validated in the development of kinase inhibitors that have improved drug-like properties and have potential for further development as anti-tumor therapeutics. This strategy is exemplified in the development of non-ATP inhibitors of cell cycle CDKs and involves as follows: 1) obtaining 3D structural information on the interactions of HAKRRLIF/RRLIF with the cyclin binding groove; 2) determining the important binding determinants for peptide interaction; 3) truncation of the peptide N-terminus containing one or more binding determinants; 4) computational identification of potential small molecule alternatives (partial ligand alternatives, PLAs) for the truncated portion of the peptide and which retain key interactions of the parent peptide; 5) synthesis or commercial sourcing of PLAs predicted to bind avidly with the sub site previously occupied by the deleted peptide residue(s); 6) synthesis of FLIPs through ligation of the best PLAs to the truncated peptide using solid phase synthesis; 7) testing of FLIPs in an in vitro binding or functional assay (fluorescence polarization in the CDK/cyclin context) followed by further characterization in a cell viability assay. A schematic representation of REPLACE strategy is shown in Figure 1. In this article, iterations of the REPLACE strategy are discussed and the application to CDK2/cyclin A described in detail. CDKs are believed to be directly or indirectly deregulated in the majority of tumors and are therefore considered appropriate cancer drug targets7. CDKs require association with cyclins for full activation and subsequently phosphorylate key proteins involved in cell cycle regulation8. The two major groups of CDKs are the isotypes that control cell cycle checkpoints [G1/S (CDK4/Cyclin D, CDK6/cyclin D and CDK4/cyclin E), S phase (CDK2/cyclin A) and G2/M (CDK1/cyclin B)] and the regulators of RNA polymerase through phosphorylation (CDK7/cyclin H, CDK8/cyclin C, CDK9/cyclin T). A key step in S phase progression occurs when the E2F1 transcription factor forms a complex with the DP protein which then binds to DNA and initiates gene transcription. CDK2/cyclin A is required to neutralize E2F1 transcriptional activity through phosphorylation thereby leading to release of the E2F1-DP complex and its subsequent degradation. Inhibition of CDK2/cyclin A is believed to maintain E2F1 in its DNA bound state leading to persistent activation. The resultant level of E2F-1 activity will surpass the threshold required to induce p53 independent apoptosis therefore suggesting a therapeutic strategy. Due to deregulated p53 and pRb pathways, high levels of E2F-1 frequently occur in cancer cells and inhibition of CDK2/cyclin A should lead to selective apoptosis in tumors and can be considered as a validated cancer target7.
Clinically investigated CDK inhibitors target the highly conserved ATP binding site leading to cross reactivity among the greater than 500 protein kinases in the human kinome and potentially giving rise to side effects and toxicity9. An alternate approach is non-ATP competitive inhibition by targeting substrate recruitment through the CBG present on cyclin positive regulatory subunit and which is therefore distinct and distant from ATP binding site10,11. The CBG is primarily a hydrophobic groove present in cyclin A, cyclin D and cyclin E and has been shown to recognize a consensus sequence found in substrates and tumor suppressors. As an isolated peptide, the cyclin binding motif (CBM) binds to the CBG and has been shown to inhibit kinase activity of the cell cycle CDKs. The CBM has been optimized to an octapeptide (HAKRRLIF, CDK2/cyclin A IC50 0.07&#177;0.02 &#181;M , CDK4/cyclin D, IC50 0.88&#177;0.34 &#181;M) and furthermore truncated to a pentapeptide representing a good compromise between molecular weight for drug-likeness and potency (RRLIF, CDK2/cyclin A IC50 1.01&#177;0.17 &#181;M, CDK4/cyclin D, IC50 25.12&#177;2.97 &#181;M)12,13. The CBGs consist of a large primary and smaller secondary hydrophobic pocket which are bridged by an acidic region (includes Glu220, Glu224 and Asp283). The key binding determinants of HAKRRLIF include the interaction of Ala2 with the secondary hydrophobic pocket, ion pairing and hydrogen bonds of Lys3, Arg 4 and Arg5 with the acidic region and a high degree of complementarity of Leu6 and Phe8 with the primary lipophilic site. In addition, numerous hydrogen bonds are contributed from the peptide backbone while Ile7 acts as a spacer residue allowing optimal contact with the primary pocket. The binding mode and interactions of HAKRRLIF with CBG is shown in Figure 2.
Targeting the CBM/CBG protein-protein interaction will inhibit kinase activity of CDK2/cyclin A, CDK2/cyclin E &#38; CDK4/cyclin D and this should trigger E2F1 mediated apoptosis of cancer cells while not affecting normal cells7. Although CBM derived peptides are effective inhibitors of cell cycle CDKs, it is unlikely that they will be useful as drugs due to their metabolic instability and general lack of cell permeability. To this end, we have applied the REPLACE strategy in order to convert these potent peptidic inhibitors into more drug-like compounds for further development of anti-tumor therapeutics exploiting deregulated E2F1 through CDK2/cyclin A inhibition. The following protocol summarizes work that has been completed in the application of REPLACE to the cyclin groove. In the first instance, drug-like capping group replacements for the N-terminal tetrapeptide of HAKRRLIF were identified. Furthermore improvements in these groups were investigated in an additional validation study for REPLACE. Representative results from these studies are also presented.

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			<td>Computational Chemistry</td>
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			<td>Accelyrs Discovery studio 3.0</td>
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			<td>Dell Optiplex Workstations</td>
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			<td>Synthetic Organic Chemistry</td>
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			<td>Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6mm, 5&#956;m column (Phenomenox Luna) for purity determination, 1H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI).</td>
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			<td>Flourescence Polarization Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>384 micro well plates, ,&#160; Micro pipets,&#160;</td>
			<td>Grenier Bio-one</td>
			<td>110256602</td>
			<td></td>
		</tr>
		<tr>
			<td>CDK4D1 and CDK2CA (well purified recombinant human kinase complex)</td>
			<td>BPS Bio Sciences</td>
			<td>40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)</td>
			<td></td>
		</tr>
		<tr>
			<td>assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1mM dithiothretiol (DTT),</td>
			<td></td>
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			<td></td>
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			<td>25 nM HEPES</td>
			<td>CALBIOCHEM</td>
			<td>375368</td>
			<td></td>
		</tr>
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			<td>NaCl</td>
			<td>Fisher</td>
			<td>127838</td>
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		<tr>
			<td>Nonidet P-40</td>
			<td>US Biological</td>
			<td>N3500</td>
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			<td>DTT</td>
			<td>Aldrich</td>
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			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
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			<td></td>
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		<tr>
			<td>DTX880 multimode detector&#160; fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein</td>
			<td>Beckman Coulter, Brea, CA</td>
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			<td>Cell Culture</td>
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			<td>96 well plates, &#160;&#160;</td>
			<td>Fisher</td>
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			<td>Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines</td>
			<td>ATCC</td>
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			<td>NU serum, DMEM media, trypsin, PEN/STRIP,&#160; MTT reagent,</td>
			<td colspan="2">Fisher, Life technology, Alfa Aesar</td>
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		<tr>
			<td>&#160;Heamocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo electron corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>5804 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator 4-8C</td>
			<td>Isotemp Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DTX880 multimode detector fitted with 595nm filter.</td>
			<td>Beckman Coulter, Brea, CA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

The interactions of HAKRRLIF with the cyclin groove are shown in Figure 2. The peptide residues that represent the key binding determinants include Ala2, Arg4, Leu6 and Phe8 with other residues providing smaller contributions12,13,18. In this case study the REPLACE strategy has been utilized in order to find fragment alternatives for residues in the N-terminal tetrapeptide of HAKRRLIF, primarily mimicking the interactions of Ala2 and Arg4. A library of potential Ncap fragments (Table 1...

Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

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