Targeting the undruggable: discovery of c-Myc oncogene small molecule folding inducers
COLLABORATORS WANTED! contact anton at q-mol.com
Keywords: oncogene, cancer, cMyc, c-MYC, undruggable, intrinsically disordered protein, transcription factor, cancer driver, lymphomas, leukemias, glioblastoma, GBM, acute myeloid leukemia, AML, cancer stem cells, Sildenafil (Viagra), epigallocatechin, theaflavins, oxidized form of ascorbic acid, dehydroascorbic acid, folding inducers, ligand-assisted protein folding, human metabolites, NCI DTP library
Summary
Q-MOL software was used to detect possible allosteric sites, followed by protein-ligand docking of NCI DTP and Human Metabolites libraries.
- Q-MOL identified several small molecule ligands from NCI DTP collection that were validated in cell-based assays and in thermal melt protein assay
- Virtual ligand screening of Human Metabolites library identified Sildenafil (Viagra), epigallocatechin, theaflavins as c-MYC binders. The predicted binding of these ligands correlates with existing published data
- Oxidized form of ascorbic acid (dehydroascorbic acid, DHA) was identified as a highly possible c-MYC binder. Many of published data suggest that oxidized form of ascorbic acid is a possible master regulator of the processes associated with proliferation and cell death.
The cMyc oncogene is the master regulator of cancer cell metabolism. The c-Myc is believed to drive over 70% of cancers, and is considered by many as Holy Grale of cancer drug development.
However, decades of efforts have failed to identify a c-Myc targeted therapeutics. cMyc protein is intrinsically disordered protein (acquires structure only when bound to one of its many binding partners), and lacks structural features such as well-defined binding pocket, believed by mainstream industry to be absolutely required for successful small molecule targeting.
c-Myc was one of the first proteins that was targeted using Q-MOL platform as part of its feature validation.
The following is a brief outline of research completed (unpublished) toward identification of cMyc-targeted therapeutics. The research was completed in collaboration with Dr. Peter Vogt, The Scripps Research Institute.
The steps below outline the basic drug discovery Q-MOL workflow as applied to c-Myc:
- Detect allosteric binding sites by scanning the molecular surface of a protein target using as docking probes known validated ligands or individual amino acids treated as small molecules
- Select docking site, dock diverse ligand library, select hits and validate them biologically. This also validates docking site selection, docking setup and overall targeting strategy
- Change chemical space of discovered active ligands by simply changing source docking ligand library
Refer to Q-MOL platform preprint for methodology details.
1) Targeting strategy: identify ligands that bind cMyc directly and induce efficient ligand-assisted folding of disordered cMyc protein into non-productive cMyc-ligand complex
Benefits of the stragegy:
thermodynamically favorable event
not subject to disengagement as part of bigger complex
virtually irreversible binding
First-in-class and validated small molecule compounds (JACS paper 2009), directly interacting with cMyc, were used as docking probes to systematically scan the molecular surface of cMyc chain (chain A) from 1NKP (bHLHZ transactivation domains of Myc/Max complex) to confirm their binding sites and to validate the Q-MOL software applicability:
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The figures above demonstrate correct Q-MOL-predicted assignment of cMyc amino acid stretches experimentally shown to interact with cMyc small molecule ligands (10009-G9, 10031-B8, 10058-F4). The Sorrento Therapeutics* ligand is predicted to interact with a different region of cMyc.
*see Sorrento Therapeutics patent (abandoned).
The cMyc molecular surface was also scanned using structures of individual amino acids as docking probes to identify general allosteric sites:
Q-MOL UI screenshots are shown. The surface scan traces of the most specific amino acid docking probes are shown as spheres. The spheres depict probabilities of probes binding: blue/small – low, red/big – large.
The docking site was selected as the area with the strongest signal, which also aligns with predictions for JACS paper ligands. The Myc/Max interface site matches that of predicted for Sorrento Therapeutics ligand, which was discovered by a binding assay that uses Myc/Max complex disruption as a read out.
The Q-MOL protein-ligand docking of diverse library containing 270K compounds against the selected docking site (Max was removed from simulation), followed by a few rounds of computational optimization of initial hits, resulted in identification of specific cMyc ligands that are potent in cells:
The data are Western blot for NDRG1 gene product that is upregulated when cMyc is downregulated. In our case, cMyc is trapped in a non-productive stable complex with a small molecule ligand causing increased expression of NDRG1 (upregulated).
Note that Sorrento Therapeutics compounds are virtually non-active in cell-based assay, albeit having nanomolar range biochemical assay IC50s as reported in the patent publication.
The ligand-assisted folding of cMyc was also directly confirmed using recombinant cMyc protein. The plot below represents corresponding thermal melt experiments:
The data represent thermal melt of cMyc protein in complex with ligand #94. The recombinant cMyc protein (the transactivation domain) was purified as inclusion bodies and dissolved in 6M urea. The refolding was initiated by diluting denatured cMyc into a buffer containing indicated concentrations of ligand #94.
2) The docking of Human Metabolites compounds library (9K compounds)
The Human Metabolites compounds library is the collection of small molecule ligands identified in human bodily fluids. These compounds represent drugs and their metabolites, biochemical metabolites, natural products. The screening of this library allows identifying compounds that are by default “approved” for human consumption as they are already found in body circulation.
The Q-MOL docking curve of the Human Metabolites compounds library:
The plot displays first 500 lowest (best) energy hits out of 9000 compounds. Note that only first 250 compounds have meaningful energy values, several leading compounds are selected (red dots).
The screenshot of Q-MOL chemical display table of the first 15 leading compounds:
The four structures are disclosed here: dehydroascorbic acid (oxidized form, rank #10), epigallocatechin sulfate (rank #12), sildenafil (Viagra) (rank #13) and theaflavin-3-gallate (rank #15).
Epigallocatechin sulfate is the metabolite of epigallocatechin gallate (EGCG) which is the primary polyphenol in green tea. Theaflavin-3-gallate is the primary polyphenol in black tea.
Recently, EGCC was shown to interact directly with cMyc transactivation domain:
“A druggable conformational switch in the c-MYC transactivation domain” Nature Communications
The binding was experimentally confirmed using mass spectrometry:
All together the above data open up a totally new facet of mechanism of cancer prevention by tea polyphenols.
Sildenafil (Viagra) represents a curios case of repurposing a well known drug. There were several reports of its activity against certain cancers and ability to enhance the effectiveness of certain chemotherapy drugs. Very recently, it was reported to interfere with cMyc activity preventing gastric cancer growth: “Repurposing of phosphodiesterase-5 inhibitor sildenafil as a therapeutic agent to prevent gastric cancer growth through suppressing c-MYC stability for IL-6 transcription” Nature Communications.
And finally, oxidized form of ascorbic acid (dehydroascorbic acid, DHA) with docking rank #10, it is a safe and widely available, in pure form, compound. Many published data suggest that oxidized form of ascorbic acid is a possible master regulator of the processes associated with proliferation and cell death. For review, see Two Distinct Faces of Vitamin C: AA vs. DHA.
c-MYC oncogene drives up to 70% of most aggressive cancers including lymphomas, leukemias, glioblastoma (GBM), acute myeloid leukemia (AML), and many other cancers originating from cancer stem cells.
What if most common and aggressive cancers could be at least prevented by simply consuming, as a dietary supplement, a safe and very accessible natural product?
Anton Cheltsov PhD