Development of Novel Drugs Targeting Chaperones of Oncogenic K-Ras

Farid Ahmad Siddiqui

Research output: Types of ThesisDoctoral ThesisCollection of Articles

Abstract

KRAS mutations account globally for about one million deaths per year, but there is as yet no approved drug against KRAS. K-Ras proteins are organized into di-/oligomeric nanoscale signaling complexes, known as nanoclusters on the plasma membrane. In this thesis, I describe two targeting approaches to indirectly inhibit the oncogenic activity of K-Ras. First, we designed inhibitors that block the interaction between the trafficking chaperone PDE6D and K-Ras, thus blocking membrane localization of K-Ras. Secondly, we showed how the major folding chaperone Hsp90 and its co-chaperone Cdc37 affect K-Ras signaling. We then went on to develop and test novel inhibitors against the interface between Hsp90 and Cdc37.

K-Ras requires several post-translational modifications, such as farnesylation, for proper organization on the plasma membrane. Interaction of farnesylated KRas with the trafficking chaperone PDE6D results in its effective solubilization and translocation in the cytosol. Available PDE6D inhibitors are easily dislodged from PDE6D through the GTP-ARL2 unloading mechanism, consequently limiting the overall cellular potency of these inhibitors. To address this problem, we improved the compound design to withstand the ejection mechanism and added a cell penetration group to increase the bioavailability.

The second part connects to our previous finding, showing that the inhibition of Hsp90/Cdc37 by conglobatin A selectively blocks the activity of K-Ras but not H-Ras, and inhibits stemness activity of cancer cells. However, the exact mechanism by which Hsp90/Cdc37 inhibition blocks the activity of K-Ras remained unclear. Here, we discovered that the inhibition of Hsp90 downregulates the K-Ras nanocluster modulator galectin-3 by inhibiting HIF-1α. Decreased expression levels of galectin-3 and Hsp90-clients B- and C-Raf jointly contributed to selectively disrupt K-Ras membrane nanoclusters, thus blocking the oncogenic activity of K-Ras.

In order to identify novel Hsp90/Cdc37 inhibitors, we first selected compounds from a computational screening and then validated their ability to interrupt the Hsp90/Cdc37 complex in a split Renilla luciferase assay. Finally, we discovered two compounds that inhibited the Hsp90/Cdc37 complex formation. By assessing these compounds in cellular assays, we confirmed their K-Ras membrane organization disrupting activity and the impairment of the signaling pathways downstream of K-Ras. Furthermore, these compounds also decreased K-Ras dependent cancer cell proliferation in 2D monolayers, 3D spheroid growth and microtumor formation. Taken together, the work of this thesis has led to the development and characterization of novel small molecule inhibitors that indirectly target K-Ras. Our findings may form the basis for the development of future therapeutic agents against K-Ras dependent human diseases.
Original languageEnglish
Supervisors/Advisors
  • Abankwa, Daniel, Supervisor
Place of PublicationÅbo
Publisher
Print ISBNs978-952-12-4031-7
Electronic ISBNs978-952-12-4032-4
Publication statusPublished - 2021
MoE publication typeG5 Doctoral dissertation (article)

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