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

Summary

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.

Abstract

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.

Introduction

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 described^1-3. 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 m....

Protocol

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.

1. Validation of the LigandFit docking protocol¹⁴

Note: In previous studies, the docking method (LigandFit¹⁵, a module in the molecular modeling program suite, Discovery Studio 3.0) was validated to ensure that this al....

Results

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 contributions^12,13,18. 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.......

Discussion

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’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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Computational Chemistry
Accelyrs Discovery studio 3.0
Dell Optiplex Workstations
Synthetic Organic Chemistry
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 4.6 mm, 5 μm 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).
Flourescence Polarization Assay
384 micro well plates, Micro pipets	Grenier Bio-one	110256602
CDK4D1 and CDK2CA (well purified recombinant human kinase complex)	BPS Bio Sciences	40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)
assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1 mM dithiothretiol (DTT))
25 nM HEPES	CALBIOCHEM	375368
NaCl	Fisher	127838
Nonidet P-40	US Biological	N3500
DTT	Aldrich
-70 °C freezer	Revco (Ultima II)
DTX880 multimode detector fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein	Beckman Coulter, Brea, CA
Cell Culture
96 well plates	Fisher
Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines	ATCC
NU serum, DMEM media, trypsin, PEN/STRIP, MTT reagent	Fisher, Life technology, Alfa Aesar
Heamocytometer	VWR
-70 °C freezer	Revco (Ultima II)
Incubator	Thermo electron corporation
Centrifuge	Eppendorf	5804 R
Refrigerator 4-8 °C	Isotemp Fisher
DTX880 multimode detector fitted with 595 nm filter	Beckman Coulter, Brea, CA