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Drug discovery

 Drug discovery is a whole process from the identification of compound synthesis, candidates, charact-erization, and assays for therapeutic efficacy to identify a compound as a potential drug. It needs the convergence of various scientific research areas, including biology, chemistry, and pharmacology.

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 Our lab is working for finding the novel drug candidate. In addition to the kinase inhibitors, we are trying to discover the promising candidate molecules based on the Protein-protein Interaction (PPI) inhibition and Targeted Protein Degradation (TPD) technology.

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Degraders

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  Identification of kinase degraders based on the PROTAC technology

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  A proteolysis-targeting chimera (PROTAC) is an emerging drug discovery platform that degrades disease-causing proteins through ubiquitin-proteasome system in cells. PROTACs are heterobifunctional molecules consisted of three parts: an E3 ligase binder, a warhead recognizing target proteins, and a linker. Once PROTACs form stable ternary complex (Target:PROTAC:E3 ligase) in cells, E2/E3 complex transfers a ubiquitin to target proteins. Subsequently, the ubiquitinated targets are recognized by proteasome where they are subjected to proteasomal degradation.

 Owing to initial success of PROTACs to target key disease proteins including FLT3, androgen receptor (AR), and BRD4, a rapid growth in researches regarding PROTACs has been achieved.

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 There are some reasons why PROTAC mediated Target protein degradation is in the limelight;

  1. Improve specificity; even when PROTAC has relatively low affinity with POI than conventional inhibitors.

  2. PROTACs have catalytic nature; this nature can achieve degradation at lower dose and reduce the off-target toxicity.

  3. It is possible to reduce the number of administrations because of the recovery time of the degraded POI.

  4. PROTAC can degrade POI by combining with not only active-site but the other multi-binding site of POI.

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With the help of PROTACs as a drug discovery platform, we have set our goals to surmount current limits of conventional kinase inhibitors particularly: a) acquiring high target selectivity, b) overcoming foregoing drug resistance, and c) targeting undruggable proteins.

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Our current interests in PROTAC research program are namely:

  1. Identification of novel E3 ligase binders forming a stable ternary complex.

  2. Rational design and development of PROTACs aiming at undruggable targets.

  3. Overcoming drug resistance arising from kinase mutations of POI by using PROTACs.

  Synthesis and structure-activity relationships of targeted protein degraders for the understudied kinase NEK9

  (Curr. Res. Chem. Biol. 2021, 1, 100008.)

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  • Nek9 is a member of the understudied Nek family of dark kinases, and constitutive activation of Nek9 is executing various cellular functions, including cancer progression, cancer proliferation.

  • Currently, there is no small molecule that is selective for Nek9.

  • We conducted the SAR study or potent degraders of the NEK9 kinase and identified the several attractive leads for further development. 

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  Novel E3 ligase binder

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  • Given the importance of factors like ligand availability and subcellular localization in determining PROTAC effectiveness, E3 ligases are expected to play a crucial role in TPD (Target Protein Degradation).

  • Despite the considerable therapeutic promise offered by PROTAC, only a small subset of the over 600 E3 ligases present in the human genome is currently employed in PROTAC applications.

  • A substantial part of the initial efforts on TPD has mainly focused on recruiting VHL or CRBN; however, to address the limitations of the existing CRBN or VHL E3 ligases and diversify the pool of E3 ligases, there is a current need to explore new E3 ligases for PROTAC technology.

Associated publication:

1. Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase (Cell Chem. Biol. 2023. Nov. 16;30(11):1414-1420)

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  Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase

  (Cell Chem. Biol. 2023. Nov. 16;30(11):1414-1420)

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  • Ubiquitin E3 ligases, such as the 'C-degron' subset, play a vital role in the ubiquitin-proteasome system by facilitating the degradation of substrate proteins.

  • Notably, the DesCEND (destruction via C-end degron) pathway, characterized by Cullin 2 RING E3 ligase (CRL2), utilizes exchangeable substrate receptors to recognize abnormal protein C-termini.

  • KLHDC2, identified as a tractable novel ubiquitin E3 ligase through high-throughput experiments, offers a promising platform for targeted protein degradation (TPD).

  • Based on these findings, we demonstrated the targeted protein degradation (TPD) mediated by the KLHDC2 ubiquitin E3 ligase as a means to regulate kinase activity.

  • We linked KLHDC2 substrate peptides, adopted from SelK with the di-glycine C-degron (PPPMAGG), to the promiscuous kinase inhibitor YHJ1039 using a polyethylene glycol (PEG) linker. This synthesis resulted in the lead compound KYH1872, demonstrating significant degradation of WEE1 and CDK4 at 1 uM.

  • Subsequent experiments utilizing MLN4924 rescue, HiBit, and pull-down assays validated that KLHDC2 serves as the E3 ligase responsible for the degradation of these kinase targets.

  Targetable PROTAC

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 It is essential to enhance selectivity for maximizing safety and efficacy of the drug. One of the strategies is to utilize targeting ligand, which is recognized by receptors highly expressed in diseased cells. We adopt this concept on PROTAC, named targetable PROTAC, to deliver PROTAC molecule specifically to the disease cells, not normal cells.

 

 Our targetable PROTAC are constructed of a targeting ligand linked via a self-cleavable linker to a PROTAC (therapeutic payload). The targetable PROTAC molecule undergo either i) internalization into the diseased cells by receptor, and cleavage of the targeting ligand inside the cells, or ii) enzymatic cleavage by diseased tissue. Then, subsequent self-cleavage of the linker fully activates the PROTAC.

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  Autophagy-mediated Degrader

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Autophagy can selectively remove damaged organelles, protein aggregates, or pathogens.

This strategy can be used as a protein degradation strategy by autophagy, not a protein degradation strategy by the proteasome.

 

 AUTAC

Large molecular targets such as aggregate proteins and amyloid proteins that cannot be degraded by PROTAC can be degraded by AUTAC, which undergoes lysosomal degradation. AUTAC is linked to a POI ligand and a guanine tag. The guanine tag induces POI to K63 polyubiquitination, and K63 polyubiquitinated target protein is recognized by SQSTM1/p62, an autophagy receptor, forming an autophagosome. The autophagosome fuse with a lysosome, and selective autophagy degradation is occurred.

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  • Our laboratory succeeded in securing a new tau-AUTAC (YUH0746) using AUTAC technology that utilizes lysosomal degradation.

  • It was confirmed that YUH0746 degrades tau protein in the doxycycline-inducible tau expression system. When treated with bafilomycin A1 (BafA1), one of the autophagy inhibitors, tau protein was not degraded.

  • Through colocalization of GFP-tau and LC3B, it was confirmed that tau protein was decomposed through autophagic degradation.

 Chaperon-Mediated Autophagy (CMA) degrader (CMA-degrader)

 HSC70 protein forms a complex with a protein with a KFERQ motif and induces the LAMP2A protein in the lysosome. The induced KFERQ motif protein unfolds and enters the lysosome through the LAMP2A oligomer. Degraded by lysosomal enzymes with the help of Lysosomal HSC70. Because HSC70 and LAMP2A recognize the KFERQ motif or KFERQ-like motif, selectivity is possible compared to other autophagy (J. Cell Sci., 2000). Since CMA is activated in cancer cells, the CMA-degrader strategy is likely to be effective (Mol. Med. Rep., 2021). It can be applied as a CMA-degrader by connecting the warhead that binds to the target protein to the KFERQ motif.

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