Nurix Therapeutics, Inc. · NASDAQ:NRIX
Nurix Therapeutics — Discovery on Target 2024
19 slides from Nurix Therapeutics's conference presentation, September 2024
Slide 1: Rational Discovery of a Small Molecule Intramolecular Glue Inhibitor of CBL-B
Rational Discovery of a Small Molecule Intramolecular Glue Inhibitor of CBL-B that Enhances T-cell Function Stefan Gajewski Discovery on Target 2024
Slide 2: Important Notice and Disclaimers
Important Notice and Disclaimers This presentation contains statements that relate to future events and expectations and as such constitute forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. When or if used in this presentation, the words “anticipa
Slide 3: CBL-B Is a Modulator of Immune Cell Activation
CBL-B Is a Modulator of Immune Cell Activation • CBL-B is an E3 ubiquitin ligase highly expressed in cells of the immune system • CBL-B regulates T, B, and NK cell activation APC CBL-B inhibition • Blocking CBL-B removes a brake on the immune MHC CD80/86 IL-2 production system Signal 1 TCR CD28 Sign
Slide 4: CBL-B Is a Modulator of Immune Cell Activation
CBL-B Is a Modulator of Immune Cell Activation Inactivation or deletion of CBL-B results in hyperactive T cells and inhibition of tumor growth. IL-2 secretion in KO and ligase inactive T cells ex vivo Ligase-inactive CBL-B knock-in mice inhibit tumor growth (TC-1 syngeneic model). Het Inactive Ligas
Slide 5: Active CBL-B Binds Ub-loaded E2 Ligases
Active CBL-B Binds Ub-loaded E2 Ligases “closed-state” “open-state” “active-state” (inactive, autoinhibited) (inactive, Y363 accessible) (active, pY363 binds E2-Ub) RING pY363 LHR LHR E2 Y363 pY-ZAP70 LHR Y363 kinase opening RING Ub closing phosphatase TKB TKB RING TKB (CBL-B – Nurix, (Model) (PDB:
Slide 6: Dynamics of CBL-B activation by a Tyrosine phosphorylated ZAP70 peptide
Dynamics of CBL-B activation by a Tyrosine phosphorylated ZAP70 peptide 6
Slide 7: CBL-B Phosphorylation Assay ("Closed State")
CBL-B Phosphorylation Assay (“Closed State”) CBL-B phosphorylation assay Tb • FRET based pY(363)-CBL-B Tb phosphorylation assay: o CBL-B N-terminal B B labeled with Bodipy Kinase + ATP o Terbium labeled αpY mAb o ZAP70 peptide for CBL- B open state induction Peptide counter assay o Src kinase and AT
Slide 8: E2~Ub Binding Assay ("Open State")
E2~Ub Binding Assay (“Open State”) • FRET based E2~Ub binding assay to pY(363)-CBL-B: o CBL-B N-terminal labeled with biotin and streptavidin-terbium o E2~Ub conjugate C-terminal labeled with Bodipy B Loss of FRET signal indicates: 1) PPI inhibition or Tb 2) closed state glue 8
Slide 9: Discovery of CBL-B Inhibitors via Open State HTS
Discovery of CBL-B Inhibitors via Open State HTS Test Funnel 104 hits selected from Peptide phosphorylation (% change) Phosphorylation assay phosphorylation HTS NRX-1 E2 binding ~90% “confirmed” from assay crude material DSF 14 positive in titration assays after re-purification SPR 1 compound valida
Slide 10: HTS Reveals a Singleton Hit
HTS Reveals a Singleton Hit N N CBL-B phosphorylation E2~Ub binding Peptide counter screen H 25 25 25 N % change from DMSO % change from DMSO % change from DMSO S N 0 0 0 O -25 -25 -25 -50 -50 -50 NRX-1 -75 -75 -75 racemic -100 -100 -100 mwt = 338 Phospho: IC50 = 39 µM -125 -125 -125 Ksol 280 µM 10
Slide 11: NRX-2 Is a Specific Inhibitor of CBL-B
NRX-2 Is a Specific Inhibitor of CBL-B E2~Ubbinding SPR N N phosphorylation H 25 N % change from DMSO (S) S N 0 O -25 NRX-2 -50 -75 eutomer -100 Phospho: IC50 = 34 µM -125 E2~Ub: IC50 = 21 µM 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 [Compound] μM N N H N SPR: KD = 23 µM S N Chiral SFC O NRX-1 N N
Slide 12: Binding Pose of NRX-2 CBL-B Inhibitor in X-ray Crystal Structure
Binding Pose of NRX-2 CBL-B Inhibitor in X-ray Crystal Structure G367 E365 K145 Y363 M366 F263 Y260 2Å resolution 12
Slide 13: SH3 and RING domain movement upon NRX-2 binding
• SH3 moves away from LHR • RING domain swings into a new • K145 stabilizes LHR conformation upon NRX-2 binding position without perturbing LHR with a niche-3 contact 13
Slide 14: Early SAR Evaluation Led to Identification of Tool Compound NRX-5
Early SAR Evaluation Led to Identification of Tool Compound NRX-5 N N E2~Ub binding H N phosphorylation N S N 25 % change from DMSO O 0 -25 NRX-4 -50 -75 Phospho: IC50 = 1.3 µM UbcH5B~Ub binding -100 E2~Ub: IC50 = 1.2 µM -125 Cbl-b phosphorylation SPR: KD = 1.5 µM 10 -4 10 -3 10 -2 10 -1 10 0 10 1 1
Slide 15: NRX-5 Stimulates Immunogenicity in PBMCs
NRX-5 Stimulates Immunogenicity in PBMCs Het Inactive Ligase mutant (C373A) IL-2 fold change Ligase KO EC50 (µM) Emax 40 NRX-5 Fold increase in %CD69+ 2.1 ≥ 4.0 (Normalized to DMSO) NRX-6 Fold increase in %CD25+ 1.4 ≥ 5.1 IL-2 fold change 30 IFN-у fold change 0.3 ≥ 4.7 20 IL-2 fold change N/D ≥ 24
Slide 16: Further Optimization of NRX-5
Further Optimization of NRX-5 E2~Ub binding 25 % change from DMSO 0 -25 -50 Headgroup Spacer -75 modifications modifications Me -100 O N N N N N H H -125 N N 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 F 3C N S N N N O O [Compound] μM NRX-5 NRX-7 Phospho: IC50 = 0.4 µM Phospho: IC50 = n/a E2~Ub: IC5
Slide 17: NRX-7 Enhances in vivo T-Cell Stimulation and Anti-Tumor Efficacy
NRX-7 Enhances in vivo T-Cell Stimulation and Anti-Tumor Efficacy Increase of CD69 and CD25 in CD4+ / CD8+ T cells 24 after CD3 stimulation – NRX-7 was administered orally before CD3 injection ✱✱ ✱ ✱✱ ✱ ✱ 100 80 100 100 ✱✱ ✱ (% of CD4+ T cells) (% of CD8+ T cells) (% of CD4+ T cells) (% of CD8+ T ce
Slide 18: Conclusions
Conclusions • CBL-B is a well-characterized E3 ligase that functions to modulate immune cell activity • We identified the autoinhibited state of CBL-B as the relevant target form for drug discovery and developed two orthogonal assays monitoring the sequential activation of this target protein • A si
Slide 19: Thank You !
Thank You ! 19