Dual Inhibitor Project

Stereogram of the structure of drug lead 1 that can be viewed by convergent viewing.

Prototypical chemotherapeutic agents for cancer treatment are based on the concept of “one-drug, one-target, one-disease”, but most forms of cancer have the ability to develop resistance mutations or are able to up-regulate targeted proteins to elude treatment with a single therapeutic agent. Polypharmacology, previously considered undesirable for a drug, has re-emerged as an approach to cancer therapy by acting on multiple targets or cellular pathways.1 Anticancer drugs displaying polypharmacology are thought to evade drug resistance due to the need for cells to become resistant across multiple pathways through simultaneous protein/enzyme mutations.
The Romo Research Group had previously synthesized both orlistat (tetrahydrolipstatin) derivatives2 as inhibitors of the thioesterase domain of fatty acid synthase (FAS-TE) and also belactosin C and derivatives as proteasome inhibitors.3 The structural similarities of belactosin and orlistat led to a hypothesis that hybrid structures of these B-lactone-containing may be capable of inhibiting both the proteasome and FAS-TE. This hypothesis was pursued by the TAMU Undergraduate MiniPharma Team and led to the synthesis and testing of >10 new hybrid inhibitors.


The in vitro enzyme assay proved their potency as inhibitors of the proteasome and FAS-TE. Both assays measure the rate of hydrolysis of fluorogenic substrates in the presence and absence of inhibitors. The most potent dual inhibitor 1 exhibited an IC50 value of 1.50 ± 0.28 uM against FAS-TE, which was 3X more potent compared to orlistat (IC50 4.63 ± 1.49 uM), while also having an IC50 of 0.37 ± 0.01 uM against the proteasome with high selectivity for ChT-L sites. (IC50 represents the concentration of a drug that is required for 50% inhibition in vitro). The ChT-L site has been the main focus for the design of antineoplastic therapeutics.

The top five potent analogs 1-5 were selected as drug leads to assess their cytotoxicity toward HeLa cells. All analogs showed IC50 values in the 10–60 uM range. In addition, an activity-based protein profiling (ABPP) experiment with the proteome of HeLa cells using an activity-based probe 6 confirmed the ability of these dual inhibitors to target both FAS and the proteasome in HeLa cells.

The Dual Inhibitor project was one of three projects undertaken by the Texas A&M Undergraduate Minipharma Program initiated by Prof. Romo. Upon his move to Baylor in Fall 2015, Prof. Romo restarted this program as the Baylor Undergraduate MiniPharma Program in Summer 2016. The TAMU MiniPharmers were involved in the following areas: (1) synthesizing inhibitors or cellular probes, (2) serum stability studies of the dual inhibitors. (3) molecular modeling and docking to develop hypotheses for new compounds to synthesize. 4


1. Anighoro, A., Bajorath, J., and Rastelli, G. Polypharmacology: Challenges and Opportunities in Drug Discovery. J. Med. Chem. 57, 7874-7887 (2014).
2. Ma, G., Zancanella, M., Oyola, Y., Richardson, R. D., Smith, J. W., and Romo, D. Total synthesis and comparative analysis of orlistat, valilactone, and a transposed orlistat derivative: Inhibitors of fatty acid synthase. Org. Lett. 8, 4497-4500 (2006).
3. (a) Cho, S. W., and Romo, D. Total synthesis of (-)-belactosin C and derivatives via double diastereoselective tandem mukaiyama aldol lactonizations. Org. Lett. 9, 1537-1540 (2007); (b) Asai, A., Hasegawa, A., Ochiai, K., Yamashita, Y., and Mizukami, T. Belactosin A, a novel antitumor antibiotic acting on cyclin/CDK mediated cell cycle regulation, produced by Streptomyces sp. J. Antibiot. (Tokyo) 53, 81-83 (2000).
4. Zhu, M., Harshbarger, W. D., Robles, O., Krysiak, J., Hull, K. G., Cho, S. W., Richardson, R. D., Yang, Y., Garcia, A.*, Spiegelman, L.*, Ramirez, B.*, Wilson, C. T.*, Yau, J. A.*, Moore, J. T.*, Walker, C. B.*, Sacchettini, J. C., Liu, W. R., Sieber, S. A., Smith, J. W., and Romo, D. A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids. Biorg. Med. Chem. 25, 2901-2916 (2017).
*Denotes TAMU Undergraduate MiniPharmers