Smitha Kizhake

Isatin Derived Spirocyclic Analogues with α-Methylene-γ-butyrolactone as Anticancer Agents: A Structure–Activity Relationship Study

Design, synthesis, and evaluation of α-methylene-γ-butyrolactone analogues and their evaluation as anticancer agents is described. SAR identified a spirocyclic analogue 19 that inhibited TNFα-induced NF-κB activity, cancer cell growth and tumor growth in an ovarian cancer model. A second iteration of synthesis and screening identified 29 which inhibited cancer cell growth with low-μM potency. Our data suggest that an isatin-derived spirocyclic α-methylene-γ-butyrolactone is a suitable core for optimization to identify novel anticancer agents.

Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1

The C-terminal domain of BRCA1(BRCT) is involved in the DNA repair pathway by recognizing the pSXXF motif in interacting proteins. It has been reported that short peptides containing this motif bind to BRCA1(BRCT) in the micromolar range with high specificity. In this work, the binding of pSXXF peptides has been studied computationally and experimentally in order to characterize their interaction with BRCA1(BRCT). Elucidation of the contacts that drive the protein–ligand interaction is critical for the development of high affinity small-molecule BRCA1 inhibitors. Molecular dynamics (MD) simulations revealed the key role of threonine at the peptide P+2 position in providing structural rigidity to the ligand in the bound state. The mutation at P+1 had minor effects. Peptide extension at the N-terminal position with the naphthyl amino acid exhibited a modest increase in binding affinity, what could be explained by the dispersion interaction of the naphthyl side-chain with a hydrophobic patch. Three in silico end-point methods were considered for the calculation of binding free energy. The Molecular Mechanics Poisson–Boltzmann Surface Area and the Solvated Interaction Energy methods gave reasonable agreement with experimental data, exhibiting a Pearlman predictive index of 0.71 and 0.78, respectively. The MM-quantum mechanics-surface area method yielded improved results, which was characterized by a Pearlman index of 0.78. The correlation coefficients were 0.59, 0.61 and 0.69, respectively. The ability to apply a QM level of theory within an end-point binding free energy protocol may provide a way for a consistent improvement of accuracy in computer-aided drug design.

Structural characterization of BRCT-tetrapeptide binding interactions.

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry. We show that BRCT is dynamic and becomes structured on binding, that pSer and Phe residues dictate overall binding, and that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in the BRCT(BRCA1) and tetrapeptides. These results provide critical insights for designing high-affinity BRCT(BRCA1) inhibitors.

Structure Activity Relationship Studies to Probe the Phosphoprotein Binding Site on the Carboxy Terminal Domains of the Breast Cancer Susceptibility Gene 1

Carboxy terminal BRCT domains of the breast cancer susceptibility gene 1 (BRCA1) bind to phosphorylated proteins through a pSXXF consensus recognition motif. We report a systematic structure-activity relationship study that maps the BRCT(BRCA1)-pSXXF binding interface, leading to identification of peptides with nanomolar binding affinities comparable to those of the previously reported 13-mer peptides and providing a clear description of the pSXXF-BRCT interface, which is essential for developing small molecule inhibitors via the peptidomimetic approach.

The paradox of conformational constraint in the design of Cbl(TKB)-binding peptides.

Solving the crystal structure of Cbl(TKB) in complex with a pentapeptide, pYTPEP, revealed that the PEP region adopted a poly-L-proline type II (PPII) helix. An unnatural amino acid termed a proline-templated glutamic acid (ptE) that constrained both the backbone and sidechain to the bound conformation was synthesized and incorporated into the pYTPXP peptide. We estimated imposing structural constraints onto the backbone and sidechain of the peptide and preorganize it to the bound conformation in solution will yield nearly an order of magnitude improvement in activity. NMR studies confirmed that the ptE-containing peptide adopts the PPII conformation, however, competitive binding studies showed an order of magnitude loss of activity. Given the emphasis that is placed on imposing structural constraints, we provide an example to support the contrary. These results point to conformational flexibility at the interface, which have implications in the design of potent Cbl(TKB)-binding peptides.

Peptide truncation leads to a twist and an unusual increase in affinity for casitas B-lineage lymphoma tyrosine kinase binding domain.

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.

Characterization of Promiscuous Binding of Phosphor Ligands to Breast-Cancer-Gene 1 (BRCA1) C-Terminal (BRCT): Molecular Dynamics, Free Energy, Entropy and Inhibitor Design

Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain. Several studies identified short phosphopeptides as tight BRCT binders. Here we investigated the thermodynamic properties of 18 phosphopeptides or peptide with phosphate mimic and three compounds with phosphate groups binding to BRCT to understand promiscuous molecular recognition and guide inhibitor design. We performed molecular dynamics (MD) simulations to investigate the interactions between inhibitors and BRCT and their dynamic behavior in the free and bound states. MD simulations revealed the key role of loops in altering the shape and size of the binding site to fit various ligands. The mining minima (M2) method was used for calculating binding free energy to explore the driving forces and the fine balance between configuration entropy loss and enthalpy gain. We designed a rigidified ligand, which showed unfavorable experimental binding affinity due to weakened enthalpy. This was because it lacked the ability to rearrange itself upon binding. Investigation of another phosphate group containing compound, C1, suggested that the entropy loss can be reduced by preventing significant narrowing of the energy well and introducing multiple new compound conformations in the bound states. From our computations, we designed an analog of C1 that introduced new intermolecular interactions to strengthen attractions while maintaining small entropic penalty. This study shows that flexible compounds do not always encounter larger entropy penalty, compared with other more rigid binders, and highlights a new strategy for inhibitor design.

Characterization of CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 – now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was ∼3.5-fold and ∼65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average >2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy.

Synthesis of aminopyrazole analogs and their evaluation as CDK inhibitors for cancer therapy

We synthesized a library of aminopyrazole analogs to systematically explore the hydrophobic pocket adjacent to the hinge region and the solvent exposed region of cyclin dependent kinases. Structure-activity relationship studies identified an optimal substitution for the hydrophobic pocket and analog 24 as a potent and selective CDK2/5 inhibitor.

Chemical Genetic Screens Identify Kinase Inhibitor Combinations that Target Anti-Apoptotic Proteins for Cancer Therapy

The study presented here provides a framework for the discovery of unique inhibitor combinations that target the apoptosis network for cancer therapy. A pair of doxycycline (Dox)-inducible cell lines that specifically report on the ability of an inhibitor to induce apoptosis by targeting either the Mcl-1 arm or the Bcl-2/Bcl-xL/Bcl-w arm were used. Cell-based assays were optimized for high throughput screening (HTS) with caspase 3/7 as a read out. HTS with a 355-member kinase inhibitor library and the panel of Dox-inducible cell lines revealed that cyclin dependent kinase (CDK) inhibitors induced apoptosis by targeting the Mcl-1 arm, whereas PI3K inhibitors induced apoptosis by targeting the Bcl-2/Bcl-xL/Bcl-w arm. Validation studies identified unique combinations that synergistically inhibited growth and induced apoptosis in a panel of cancer cell lines. Since these inhibitors have been or are currently in clinical trials as single agents, the combinations can be rapidly translated to the clinics.