poster discovery
An array of Targeted Degradation approaches offered as a partAn array of Targeted Degradation approaches offered as a part of integrated drug discovery services by o2h Discoveryof integrated drug discovery services by o2h Discovery
Prasad SulkshanePrasad Sulkshane, Romit Bose, Keith Woodley, Alice Willer, Shubham Gourana, Anagha Dahake, Animesh Pandit, Jackline Agwenge, Christian Mutti, Simon Stockwell, Rajesh Yesodharan,, Romit Bose, Keith Woodley, Alice Willer, Shubham Gourana, Anagha Dahake, Animesh Pandit, Jackline Agwenge, Christian Mutti, Simon Stockwell, Rajesh Yesodharan, Sunil ShahSunil Shah, Prashant Shah, Prashant Shahand Gayathri Sadasivamand Gayathri Sadasivam
In vitro Biology, o2h Discovery Pvt. Ltd., Changodar, Ahmedabad, India In vitro Biology, o2h Discovery Pvt. Ltd., Changodar, Ahmedabad, India In vitro Biology, o2h Discovery Pvt. Ltd., Mill SciTech Park, Hauxton, Cambridgeshire, UK In vitro Biology, o2h Discovery Pvt. Ltd., Mill SciTech Park, Hauxton, Cambridgeshire, UK *Correspondence to [email protected]*Correspondence to [email protected] o2h Discoveryo2h Discovery o2h Discovery provides early-stage drug discovery services from its advanced facilities in Ahmedabad, India, and Cambridge, UK.o2h Discovery provides early-stage drug discovery services from its advanced facilities in Ahmedabad, India, and Cambridge, UK. The company has expanded its expertise in targeted protein degradation (TPD), offering a comprehensive suite of solutions for PROTAC design, synthesis, screening, and a customizable "off-the-shelf"The company has expanded its expertise in targeted protein degradation (TPD), offering a comprehensive suite of solutions for PROTAC design, synthesis, screening, and a customizable "off-the-shelf" PROTAC toolbox. Our experienced biology team provides assays for binary and ternary complex formation, protein degradation, and DC50 estimation, ensuring fully integrated capabilities.PROTAC toolbox. Our experienced biology team provides assays for binary and ternary complex formation, protein degradation, and DC50 estimation, ensuring fully integrated capabilities. Leveraging expertise in small molecule discovery and bespoke assay development, o2h enables screening of small molecules that bind to unique RNA folds.Leveraging expertise in small molecule discovery and bespoke assay development, o2h enables screening of small molecules that bind to unique RNA folds. Drawing on insights from Tong et al. (2023), o2h Discovery is applying these findings to design RNA-targeting molecules. By conjugating RNA-binding small molecules with RNase L-recruitingDrawing on insights from Tong et al. (2023), o2h Discovery is applying these findings to design RNA-targeting molecules. By conjugating RNA-binding small molecules with RNase L-recruiting agents, or RIBOTACs, o2h aims to develop novel RNA degraders using a PROTAC-like approach, enhancing RNA-targeted drug discovery.agents, or RIBOTACs, o2h aims to develop novel RNA degraders using a PROTAC-like approach, enhancing RNA-targeted drug discovery. Looking ahead, o2h is also exploring the use of DUBTACs for protein stabilization and other TAC-based mechanisms to clear protein aggregates, broadening its scope in targeted degradationLooking ahead, o2h is also exploring the use of DUBTACs for protein stabilization and other TAC-based mechanisms to clear protein aggregates, broadening its scope in targeted degradation strategies.strategies.
PROTAC (PROteolysis TArgeting Chimera)PROTAC (PROteolysis TArgeting Chimera)
RiboTAC (RNA-TArgeting Chimera)RiboTAC (RNA-TArgeting Chimera)
Constructed to bind both targetConstructed to bind both target and E3 ligase through a crosslinkerand E3 ligase through a crosslinker
Ternary complexTernary complex
Degrader recruits target to E3 ligaseDegrader recruits target to E3 ligase
Specific questions:Specific questions:
Current RNA-targeting modalities have been limited to antisense oligonucleotides, which mediateCurrent RNA-targeting modalities have been limited to antisense oligonucleotides, which mediate Ribonuclease-dependent RNA cleavage.Ribonuclease-dependent RNA cleavage.
Q. Is my target degraded?Q. Is my target degraded?
However, RNA is remarkably structured and intricately folded as hairpin loops through itsHowever, RNA is remarkably structured and intricately folded as hairpin loops through its Q. What is the binary affinity for POI & E3 ligase?Q. What is the binary affinity for POI & E3 ligase?intramolecular base pairing, forming a series of unique secondary structures.intramolecular base pairing, forming a series of unique secondary structures. Q. Do they form ternary complex?Q. Do they form ternary complex? Q. Is my target ubiquitinated?Q. Is my target ubiquitinated?
These RNA folds, similar to motifs or domains in proteins, may offer a unique opportunityThese RNA folds, similar to motifs or domains in proteins, may offer a unique opportunity for targeting by small molecules through specific binding.for targeting by small molecules through specific binding.
PolyubiquitinationPolyubiquitination
Ubiquitin (Ub) added to target’sUbiquitin (Ub) added to target’s Lys residues
Lys residues
Target degradationTarget degradation
Ub-marked target is degradedUb-marked target is degraded by the proteasomeby the proteasome
Case Study:Case Study: Target protein:Target protein:BD2 domain of BRD4BD2 domain of BRD4 E3 Ubiquitin ligase:E3 Ubiquitin ligase:VHL
The biological outcome of such RNA binding small molecules can be further enhanced byThe biological outcome of such RNA binding small molecules can be further enhanced by appending a functional group to it, which can recruit a Ribonuclease, activate it and cleaveappending a functional group to it, which can recruit a Ribonuclease, activate it and cleave the target RNA.the target RNA.
We have developed SPR & Fluorescence Dye Displacement assays as robust and highlyWe have developed SPR & Fluorescence Dye Displacement assays as robust and highly sensitive methods to study these specific RNA-small molecule interactions.sensitive methods to study these specific RNA-small molecule interactions.
Demonstration of ternary complex formation & target protein degradation byDemonstration of ternary complex formation & target protein degradation by Nanoluciferase assay:Nanoluciferase assay:
Studying the specific interactions between c-Jun mRNA and a small moleculeStudying the specific interactions between c-Jun mRNA and a small molecule [Ref. Tong et al. (2023)].[Ref. Tong et al. (2023)].
A specific part of c-Jun mRNA bearing a unique loop/kink – Wild Type (WT), where aA specific part of c-Jun mRNA bearing a unique loop/kink – Wild Type (WT), where a HEK293 cells expressing NanoLuc-BRD4 (Donor) and Halo-Tag-VHL (acceptor) at 1:100 ratio.HEK293 cells expressing NanoLuc-BRD4 (Donor) and Halo-Tag-VHL (acceptor) at 1:100 ratio.small molecule – c-Jun mRNA binder binds.small molecule – c-Jun mRNA binder binds.
Ternary Complex FormationTernary Complex Formation
Negative control: Mutant c-Jun mRNA, which lacks the unique kink (the binding site).Negative control: Mutant c-Jun mRNA, which lacks the unique kink (the binding site).
Unrelated small molecule: c-Myc mRNA binder.Unrelated small molecule: c-Myc mRNA binder.
Demonstration of RNA-small molecule interactions by SPR:Demonstration of RNA-small molecule interactions by SPR:
In SPR assay, the small moleculeIn SPR assay, the small molecule c-Jun binder showed specificc-Jun binder showed specific binding towards WT form ofbinding towards WT form of c-Jun mRNA as compared to itsc-Jun mRNA as compared to its mutant form, such that uponmutant form, such that upon mutant subtraction, themutant subtraction, the sensorgram appear positive.sensorgram appear positive.
The unrelated small moleculeThe unrelated small molecule c-Myc mRNA binder did notc-Myc mRNA binder did not
Demonstration of target protein (BRD4) degradation by split Nanoluciferase assay:Demonstration of target protein (BRD4) degradation by split Nanoluciferase assay:
exhibit binding to the c-Junexhibit binding to the c-Jun mRNA, with the net effectmRNA, with the net effect
In HEK293 cells stably expressing LgBitIn HEK293 cells stably expressing LgBit nanoluciferase fragment, we knocked innanoluciferase fragment, we knocked inAssessing BRD4 degradation by HiBit-LgBit assayAssessing BRD4 degradation by HiBit-LgBit assay being marginally negativebeing marginally negative sensorgrams.sensorgrams.
the corresponding HiBit fragment at thethe corresponding HiBit fragment at the endogenous locus of BRD4.endogenous locus of BRD4.
The LgBit fragment has a high affinity for theThe LgBit fragment has a high affinity for the HiBit fragment, thereby reconstituting theHiBit fragment, thereby reconstituting the active NanoBiTactive NanoBiT(Nanoluciferase) enzyme.(Nanoluciferase) enzyme.
Demonstration of RNA-small molecule interactions by Fluorescent dye displacement assayDemonstration of RNA-small molecule interactions by Fluorescent dye displacement assay
Expression of BRD4 therefore,Expression of BRD4 therefore, creates an active nanoluciferase.creates an active nanoluciferase.
Degradation of BRD4 thus causes aDegradation of BRD4 thus causes a decrease in the Nanoluciferase signal.decrease in the Nanoluciferase signal.
Addition of small Addition of small molecule binder molecule binder
Demonstration of binary and ternary complex formation by SPR:Demonstration of binary and ternary complex formation by SPR:
Fluorescence readout showing displacement of the ToPro RNA binder by the c-Jun binder. As theFluorescence readout showing displacement of the ToPro RNA binder by the c-Jun binder. As the concentration of the c-Jun binder increases, fluorescence decreases. No displacement is observedconcentration of the c-Jun binder increases, fluorescence decreases. No displacement is observed with the mutant c-Jun mRNA.with the mutant c-Jun mRNA.
Next, we plan to demonstrate the ternary complex of RNA-RiboTAC-RNaseLNext, we plan to demonstrate the ternary complex of RNA-RiboTAC-RNaseL by SPR and evaluate the RiboTAC efficacy in cellular studies.by SPR and evaluate the RiboTAC efficacy in cellular studies.
Demonstration of target proteinDemonstration of target protein degradation by western blotting:degradation by western blotting:
HeLa cells transfected withHeLa cells transfected with the Nanoluc-BRD4(BD2)the Nanoluc-BRD4(BD2) plasmid and 24 hours laterplasmid and 24 hours later treated with indicatedtreated with indicated concentrations of MZ1 for 6concentrations of MZ1 for 6 hours. The cell lysates werehours. The cell lysates were analyzed by western blottinganalyzed by western blotting for the indicated proteins.for the indicated proteins.