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Please do not hesitate to contact Dr. Adibekian by email (aadibeki[at]scripps.edu) to inquire about the newest projects in our lab!

Cysteines with enhanced nucleophilicity (so-called hyperreactive cysteines) are found in diverse families of enzymes of biomedical importance such as proteases, kinases and oxidoreductases and, intriguingly, also on protein classes that are traditionally considered ‘undruggable’. Hyperreactive cysteines can be chemoselectively modified with moderately reactive electrophilic probes such as haloacetamides and α,β-unsaturated carbonyl compounds and this creates an opportunity for the development of novel pharmacological agents capable of selectively interfering with the activity of ‘undruggable’ proteins.

Our laboratory is interested in the discovery of new cysteine-reactive small molecules with intriguing biomedical activities using chemical proteomics platforms (chemoproteomics-enabled drug discovery). We are especially interested in identification of small molecules that target functional cysteines on proteins with proven oncogenic activity that are currently considered ‘undruggable’. We have recently developed a new mass spectrometry-based chemoproteomic method that allows detection and monitoring of up to 3000 reactive cysteines in any given cellular proteome in one single experiment, thus providing a comprehensive proteome-wide picture of small molecule-cysteine interactions (Angew. Chem. Int. Ed. 2015, 54, 6057). This is achieved via strategic use of two clickable, cysteine-reactive chemical probes with complementary selectivity profiles, iodoacetamide alkyne and previously reported ethynyl benziodoxolone probe JW-RF-010 (Figure 1). We showed that JW-RF-010 alkynylates cysteines in complex proteomes fast, under mild physiological conditions, and with a very high degree of chemoselectivity. We demonstrated the utility of JW-RF-010 for proteomics by identifying the proteomic targets of curcumin, a natural product with anticancer activity. Our results indicate that curcumin covalently modifies several key players of cellular signaling, including the kinase CSNK1G. 

Alkynylation of cysteine-containing proteins with the clickable EBX probe JW-RF-010 for proteomic profiling experiments.

Figure 1. Alkynylation of cysteine-containing proteins with the clickable EBX probe JW-RF-010 for proteomic profiling experiments.

We are currently applying our cysteinome profiling method to identify molecular targets of natural products with intriguing anticancer activities with the goal of understanding the underlying mechanisms of action of these molecules. For example, we discovered that the sesquiterpene lactone deoxyelephantopin (DEP) as well as several structural analogs of this natural product covalently modify the zinc-bound Cys190 in a zinc finger motif of PPARγ (Nat. Commun. 2016, 7, 12470), thus offering a completely novel pharmacological mechanism for modulating PPARγ activity and serving as a blueprint for the development of potent, irreversible antagonists of PPARγ or other transcription factors with zinc finger motif (Figure 2). The protein targets of several other natural products with intriguing anticancer activities are currently being investigated in our laboratory. 

Left: Chemical structures of deoxyelephantopin (DEP) and the DEP analog 19a. Right: Predicted model of 19a bound to human PPARγ (right).

Figure 2. Left: Chemical structures of deoxyelephantopin (DEP) and the DEP analog 19a. Right: Predicted model of 19a bound to human PPARγ.

Furthermore, we elucidated the exact molecular mechanism of the so-called thiol-mediated uptake (J. Am. Chem. Soc. 2017, 139, 231). Thiol-mediated uptake is a novel approach that makes use of dynamic covalent interactions with cell surface cysteines to deliver hydrophilic cargos inside the cells. We showed that appendage of a single asparagusic acid residue (AspA tag) is sufficient to ensure efficient celullar uptake and intracellular distribution of fully unprotected peptides (Figure 3). For example, we induced apoptotic response in cancer cells using long (up to 20mer), AspA-modified proapoptotic BH3 domain peptides. Chemical proteomics experiments documented covalent bond formation between the AspA tag and the cysteines 556 and 558 on the surface of the transferrin receptor (TFRC) resulting in subsequent endocytic uptake of the payload. The small size, low cellular toxicity and the efficient transferrin receptor-mediated uptake render the AspA tag highly attractive for various life science applications. We are currently investigating the potential of cyclic disulfides for the cellular uptake of other important hydrophilic biomolecules and drugs.  

Top: Schematic representation of thiol-mediated uptake of AspA-modified peptides. Solvent-exposed cysteines in the extracellular domain of the transferrin receptor react with the strained disulfide ring of the asparagusic acid tag and the covalently bound cargo is taken up by the cell. Bottom: Confocal images of HeLa Kyoto cells treated with an AspA-tagged fluorescent peptide for increasing amounts of time.

Figure 3. Top: Schematic representation of thiol-mediated uptake of AspA-modified peptides. Solvent-exposed cysteines in the extracellular domain of the transferrin receptor react with the strained disulfide ring of the asparagusic acid tag and the covalently bound cargo is taken up by the cell. Bottom: Confocal images of HeLa Kyoto cells treated with an AspA-tagged fluorescent peptide for increasing amounts of time.

Finally, we are also developing novel chemical strategies that allow rapid access to collections of structurally diverse cysteine-reactive small molecules. Using our cysteinome profiling methods, we are screening these collections against series of cancer-related protein targets directly in complex cellular proteomes. For example, we prepared a collection of chroromethyl triazoles (CMTs) that are accessible from commercially available substrates in just two chemical steps. From this collection, we identified compound AA-CW236 as the first non-pseudosubstrate inhibitor of O6-alkylguanine DNA alkyltransferase (MGMT), a DNA repair protein that renders several devastating forms of cancer resistant to chemotherapy (Figure 4). Using proteomics profiling, we showed that AA-CW236 exhibits high degree of selectivity towards MGMT. We validated the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast cancer cells using fluorescence imaging (Angew. Chem. Int. Ed. 2016, 55, 2911). Our results may open a new avenue towards development of a clinically approved MGMT inhibitor. We are currently pursuing several other oncologically important targets of chloromethyl triazoles and also preparing new collections of cysteine-reactive inhibitors. 

Left: Chemical structure of the chloromethyl triazole inhibitor AA-CW236. Right: Predicted model of AA-CW236 bound to human MGMT.

Figure 4. Left: Chemical structure of the chloromethyl triazole inhibitor AA-CW236. Right: Predicted model of AA-CW236 bound to human MGMT.