Amarnath Natarajan

Anticancer activity of Celastrol in combination with ErbB2-targeted therapeutics for treatment of ErbB2-overexpressing breast cancers.

The receptor tyrosine kinase ErbB2 is overexpressed in up to a third of breast cancers, allowing targeted therapy with ErbB2-directed humanized antibodies such as Trastuzumab. Concurrent targeting of ErbB2 stability with HSP90 inhibitors is synergistic with Trastuzumab, suggesting that pharmacological agents that can inhibit HSP90 as well as signaling pathways activated by ErbB2 could be useful against ErbB2-overexpressing breast cancers. The triterpene natural product Celastrol inhibits HSP90 and several pathways relevant to ErbB2-dependent oncogenesis including the NFκB pathway and the proteasome, and has shown promising activity in other cancer models. Here, we demonstrate that Celastrol exhibits in vitro antitumor activity against a panel of human breast cancer cell lines with selectivity towards those overexpressing ErbB2. Celastrol strongly synergized with ErbB2-targeted therapeutics Trastuzumab and Lapatinib, producing higher cytotoxicity with substantially lower doses of Celastrol. Celastrol significantly retarded the rate of growth of ErbB2-overexpressing human breast cancer cells in a mouse xenograft model with only minor systemic toxicity. Mechanistically, Celastrol not only induced the expected ubiquitinylation and degradation of ErbB2 and other HSP90 client proteins, but it also increased the levels of reactive oxygen species (ROS). Our studies show that the Michael Acceptor functionality in Celastrol is important for its ability to destabilize ErbB2 and exert its bioactivity against ErbB2-overexpressing breast cancer cells. These studies suggest the potential use of Michael acceptor-containing molecules as novel therapeutic modalities against ErbB2-driven breast cancer by targeting multiple biological attributes of the driver oncogene.

Cyclin Dependent Kinase 9 Inhibitors for Cancer Therapy

Cyclin dependent kinase (CDK) inhibitors have been the topic of intense research for nearly 2 decades due to their widely varied and critical functions within the cell. Recently CDK9 has emerged as a druggable target for the development of cancer therapeutics. CDK9 plays a crucial role in transcription regulation; specifically, CDK9 mediated transcriptional regulation of short-lived antiapoptotic proteins is critical for the survival of transformed cells. Focused chemical libraries based on a plethora of scaffolds have resulted in mixed success with regard to the development of selective CDK9 inhibitors. Here we review the regulation of CDK9, its cellular functions, and common core structures used to target CDK9, along with their selectivity profile and efficacy in vitro and in vivo.

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.

Perturbing pro-survival proteins using quinoxaline derivatives: A structure activity relationship study

In HeLa cells the combinatorial knockdown of Bcl-xL and Mcl-1 is sufficient to induce spontaneous apoptosis. Quinoxaline derivatives were screened for the induction of Mcl-1 dependent apoptosis using a cell line without functional Bcl-xL. Quinoxaline urea analog 1 h was able to specifically induce apoptosis in an Mcl-1 dependent manner. We demonstrate that even small changes to 1h results in dramatic loss of activity. In addition, 1 h and ABT-737 synergistically inhibit cell growth and induce apoptosis. Our results also suggest that 1h could have therapeutic potential against ABT-737 refractory cancer.

Micellar formulation of indocyanine green for phototherapy of melanoma.

Phototherapy (PT), a light activated treatment modality, is a potential therapeutic option for the treatment of melanoma. In spite of the excellent safety profile and absorption in the near infrared (NIR) range, clinical potential of indocyanine green (ICG) as PT is limited by its short half-life and inefficient tumor accumulation. In this study, we have covalently conjugated ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling, which self-assembled into micelles with a particle size of 30-50 nm and high ICG loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiate on at 4 °C revealed photodynamic and photothermal as mechanism of cytotoxicity of ICG solution and ICG conjugated micelles, respectively. In vivo NIR imaging demonstrated that ICG conjugated micelles prolonged its circulation and increased tumor accumulation through enhanced permeability and retention (EPR) effect. Enhanced tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to ICG solution and control in a A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for PT and imaging of melanoma.

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.

Face selective reduction of the exocyclic double bond in isatin derived spirocyclic lactones

We report an unusual face selective reduction of the exocyclic double bond in the α-methylene-γ-butyrolactone motif of spiro-oxindole systems. The spiro-oxindoles were assembled by an indium metal mediated Barbier-type reaction followed by an acid catalyzed lactonization.

Synthesis and evaluation of macrocyclic diarylether heptanoid natural products and their analogs

The macrocyclic diarylether heptanoid (MDEH) natural products have been used in folk medicine for centuries. MDEHs are reported to exert anti-tumor properties by inhibiting the activation of NF-κB. Here we report the synthesis of a small MDEH library (first reported synthesis of racemic platycarynol) using a Grubbs cross metathesis/Ullmann cyclization strategy. Evaluation of the library led to the identification of MDEH 9b which sensitizes pancreatic cancer cells to gemcitabine mediated growth inhibition and apoptosis.

2,3-Substituted quinoxalin-6-amine analogs as antiproliferatives: A structure-activity relationship study

The quinoxaline core is considered a privileged scaffold as it is found in a variety of biologically relevant molecules. Here we report the synthesis of a quinoxalin-6-amine library, screening against a panel of cancer cell lines and a structure-activity relationship (SAR). This resulted in the identification of a bisfuranylquinoxalineurea analog (7c) that has low micromolar potency against the panel of cancer cell lines. We also show that cells treated with quinoxalineurea 7c results in caspase 3/7 activation, PARP cleavage and Mcl-1 dependent apoptosis.

RAC1 GTPase promotes the survival of breast cancer cells in response to hyper-fractionated radiation treatment

Radiation therapy is a staple approach for cancer treatment, whereas radioresistance of cancer cells remains a substantial clinical problem. In response to ionizing radiation (IR) induced DNA damage, cancer cells can sustain/activate pro-survival signaling pathways, leading to apoptotic resistance and induction of cell cycle checkpoint/DNA repair. Previous studies show that Rac1 GTPase is overexpressed/hyperactivated in breast cancer cells and is associated with poor prognosis. Studies from our laboratory reveal that Rac1 activity is necessary for G2/M checkpoint activation and cell survival in response to IR exposure of breast and pancreatic cancer cells. In this study, we investigated the effect of Rac1 on the survival of breast cancer cells treated with hyper-fractionated radiation (HFR), which is used clinically for cancer treatment. Results in this report indicate that Rac1 protein expression is increased in the breast cancer cells that survived HFR compared with parental cells. Furthermore, this increase of Rac1 is associated with enhanced activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signaling pathways and increased levels of anti-apoptotic protein Bcl-xL and Mcl-1, which are downstream targets of ERK1/2 and NF-κB signaling pathways. Using Rac1-specific inhibitor and dominant-negative mutant N17Rac1, here we demonstrate that Rac1 inhibition decreases the phosphorylation of ERK1/2 and inhibitory κBα (IκBα), as well as the levels of Bcl-xL and Mcl-1 protein in the HFR-selected breast cancer cells. Moreover, inhibition of Rac1 using either small molecule inhibitor or dominant-negative N17Rac1 abrogates clonogenic survival of HFR-selected breast cancer cells and decreases the level of intact poly(ADP-ribose) polymerase, which is indicative of apoptosis induction. Collectively, results in this report suggest that Rac1 signaling is essential for the survival of breast cancer cells subjected to HFR and implicate Rac1 in radioresistance of breast cancer cells. These studies also provide the basis to explore Rac1 as a therapeutic target for radioresistant breast cancer cells.