A quantitative LC-MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue.

Compound 27 {1, 12-bis[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]dodecane-1,12-dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-MS/MS) has been developed for the quantification of this compound. Liquid-liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse-phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2-200 ng/mL with the intra- and inter-day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.

Synthesis and biological evaluation of anti-cancer agents that selectively inhibit Her2 over-expressed breast cancer cell growth via down-regulation of Her2 protein.

Compound JCC76 selectively inhibited the proliferation of human epidermal growth factor 2 (Her2) over-expressed breast cancer cells. In the current study, a ligand based structural optimization was performed to generate new analogs, and we identified derivatives 16 and 17 that showed improved activity and selectivity against Her2 positive breast cancer cells. A structure activity relationship (SAR) was summarized. Compounds 16 and 17 were also examined by western blot assay to check their effect on Her2 protein. The results reveal that the compounds could decrease the Her2 protein, which explains their selectivity to Her2 over-expressed breast cancer cells. Furthermore, the compounds inhibited the chaperone activity of small chaperone protein that could stabilize Her2 protein.

Synthesis of Vorinostat and cholesterol conjugate to enhance the cancer cell uptake selectivity.

Histone deacetylase (HDAC) inhibitors modulate various cellular functions including proliferation, differentiation, and apoptosis. Vorinostat (SuberAniloHydroxamic Acid, SAHA) is the first HDAC inhibitor approved by FDA for cancer treatment. However, SAHA distributes in cancer tissue and normal tissue in similar levels. It will be ideal to selectively deliver SAHA into cancer cells. Rapidly growing cancer cells have a great need of cholesterol. Low-density lipoprotein (LDL) is the major cholesterol carrier in plasma and its uptake is mediated by LDL-receptor (LDL-R), a glycoprotein overexpressed on the surface of cancer cells. Herein, we designed and synthesized a SAHA cholesterol conjugate, and further formed the conjugate containing particles with LDL as the carrier. The diameters of the particles were determined. The inhibitory activity of the particles carrying the conjugate was determined with cancer cell proliferation assay, and the hydrolysis of the conjugate by the enzymes in cancer cells was confirmed with LC-MS/MS.

Copalic acid analogs down-regulate androgen receptor and inhibit small chaperone protein.

Copalic acid, one of the diterpenoid acids in copaiba oil, inhibited the chaperone function of α-crystallin and heat shock protein 27kD (HSP27). It also showed potent activity in decreasing an HSP27 client protein, androgen receptor (AR), which makes it useful in prostate cancer treatment or prevention. To develop potent drug candidates to decrease the AR level in prostate cancer cells, more copalic acid analogs were synthesized. Using the level of AR as the readout, 15 of the copalic acid analogs were screened and two compounds were much more potent than copalic acid. The compounds also dose-dependently inhibited AR positive prostate cancer cell growth. Furthermore, they inhibited the chaperone activity of α-crystallin as well.

Synthesis and biological evaluation of selective tubulin inhibitors as anti-trypanosomal agents.

African trypanosomiasis is still a threat to human health due to the severe side-effects of current drugs. We identified selective tubulin inhibitors that showed the promise to the treatment of this disease, which was based on the tubulin protein structural difference between mammalian and trypanosome cells. Further lead optimization was performed in the current study to improve the efficiency of the drug candidates. We used Trypanosoma brucei brucei cells as the parasite model, and human normal kidney cells and mouse macrophage cells as the host model to evaluate the compounds. One new analog showed great potency with an IC50 of 70nM to inhibit the growth of trypanosome cells and did not affect the viability of mammalian cells. Western blot analyses reveal that the compound decreased tubulin polymerization in T. brucei cells. A detailed structure activity relationship (SAR) was summarized that will be used to guide future lead optimization.

Structural optimization of non-nucleoside DNA methyltransferase inhibitor as anti-cancer agent.

Inhibition of DNA methyltransferase 1 (DNMT1) can reverse the malignant behavior of cancer cells by restoring expression of aberrantly silenced genes that are required for differentiation, senescence, and apoptosis. Clinically used DNMT1 inhibitors decitabine and azacitidine inhibit their target by covalent trapping after incorporation into DNA as azacytidine analogs. These nucleoside compounds are prone to rapid enzymatic inactivation in blood, posing challenges to the development of purely epigenetic dosing schedules. Non-nucleoside compounds that suppress expression or function of DNMT1 may overcome this problem. Using a high-throughput PCR-based site specific chromatin condensation assay, we identified a compound that reactivated Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) in myeloma cells and suppressed expression of DNMT1 from a library of 5120 chemically diverse small molecules. Lead optimization was performed to generate 26 new analogs with lung cancer proliferation and DNMT1 expression as activity readout. Two of the new derivatives showed 2 fold improvement of growth inhibiting potency and also decreased DNMT1 protein levels in lung cancer cells.

Design and synthesis of small molecule agonists of EphA2 receptor.

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure-activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor.

HMBA is a putative HSP70 activator stimulating HEXIM1 expression that is down-regulated by estrogen.

Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is identified as a novel inhibitor of estrogen stimulated breast cell growth, and it suppresses estrogen receptor-α transcriptional activity. HEXIM1 protein level has been found to be downregulated by estrogens. Recently, HEXIM1 has been found to inhibit androgen receptor transcriptional activity as well. Researchers have used Hexamethylene bis-acetamide (HMBA) for decades to stimulate HEXIM1 expression, which also inhibit estrogen stimulated breast cancer cell gene activation and androgen stimulated prostate cancer gene activation. However, the direct molecular targets of HMBA that modulate the induction of HEXIM1 expression in mammalian cells have not been identified. Based on HMBA and its more potent analog 4a1, we designed molecular probes to pull down the binding proteins of these compounds. Via proteomic approach and biological assays, we demonstrate that HMBA and 4a1 are actually heat shock protein 70 (HSP70) binders. The known HSP70 activator showed similar activity as HMBA and 4a1 to induce HEXIM1 expression, suggesting that HMBA and 4a1 might be putative HSP70 activators. Molecular target identification of HMBA and 4a1 could lead to further structural optimization of the parental compound to generate more potent derivatives to stimulate HEXIM1 expression, which could be a novel approach for hormone dependent breast cancer and prostate cancer treatment.