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AN9025 exhibits a 4-fold stronger binding affinity to CypA compared to RMC-6236, driven by its slower dissociation rate. Screening Cell Lines (n=43) $1B,&ORJBȝ0 50&B,&ORJBȝ0 100 X !"#$%&'()*+ ,#-,.,/01 234& 5!" 6,#"13720849:; 5!# 5%&'(<=! !*!$.0>#?+ @1,$A0849:; 5%&'B%&C D,E1>4/,0# %&C ! 0 2 4 6 SDQï5$6 IC50(uM)_log10 SDQí5$6 G12X G13X Q61X Wild Type $1 50& Abstract #4377 AN9025, an orally bioavailable pan-RAS(ON) inhibitor with potent, broad-spectrum anti-tumor activity 0 100 200 300 400 Time(s) Response(nm) 0 0.1 0.2 0.3 Kinetic analysis of RMC-6236 Binding to CypA KD1=12.4 nM 0 100 200 300 400 Time(s) 0 0.1 0.3 0.2 Kinetic analysis of AN9025 Binding to CypA KD1=3.2 nM Response(nm) A B Shuaishuai Liu; Meng Lv; Xiangyu Fu; Chenchen Zou; Hannah Yan; Tanya Yang; Hao YF
Feifan Li; Zhao Sun; Xiaoli Zhu; Hongyang Zhu; Meng Chen; Junying Li; Zhiyong Yu; Nanhai He Adlai Nortye Ltd, Hangzhou, China Figure 1. MOA of pan-RAS(ON)i Figure 2. Binding kinetics of AN9025 and RMC-6236 to CypA. His-tagged CypA was immo- bilized on an NTA sensor and then incubated with AN9025 (A) or RMC-6236 (B) to monitor BLI signal changes associated with binary complex formation. Figure 3. Kinetic of tri-complex formation induced by AN9025 and RMC-6236. His-tag KRASG12D protein in the GMPPNP-bound state was first immobilized on a NTA sensor, followed by incubation with with AN9025 (A) or RMC-6236 (B) and CypA to monitor the BLI signal changes indicative of tri-complex formation. Table 1. Tri-complex binding affinity of AN9025 and RMC-6236 with various RAS mutant proteins ADME A B Figure 3. Cancer cell line panel screening. A. Correlation of IC 50 values between AN9025 and RMC-6236. IC50 values of RMC-6236 (x-axis) and A9025 (y-axis) were compared across 43 cell lines with different RAS mutations. B. IC50 values of AN9025 and RMC-6236 stratified by RAS genotype. 0 10 20 30 40 0 400 800 1200 1600 2000 Days after the start of treatment Tumor Volume (mm3) HPAC (KRASG12D, PDAC) Vehicle(p.o., QD) RMC-6236(25 mg/kg, p.o., QD) RMC-6236(10 mg/kg, p.o., QD) AN9025(0.1 mg/kg, p.o., QD) Treatment Stop AN9025(0.04 mg/kg, p.o., QD) 0 10 20 30 40 -15 -10 -5 0 5 10 15 Days after the start of treatment Body Weight Change (%) HPAC (KRASG12D, PDAC) 0 5 10 15 20 25 -20 -10 0 10 Days after the start of treatment HepG2 (NRASQ61L, LIHB) Body Weight Change (%) 0 5 10 15 20 25 0 500 1000 1500 Days after the start of treatment Tumor Volume (mm3) HepG2 (NRASQ61L, LIHB) Vehicle(p.o., QD) RMC-6236(25 mg/kg, p.o., QD) RMC-6236(10 mg/kg, p.o., QD) AN9025(0.15 mg/kg, p.o., QD) AN9025(0.06 mg/kg, p.o., QD) 0 5 10 15 20 25 30 -20 -10 0 10 Days after the start of treatment HCT116 (KRASG13D, CRC) Body Weight Change (%) 0 10 20 30 0 500 1000 1500 2000 2500 Days after the start of treatment Tumor Volume (mm3) HCT116 (KRASG13D, CRC) Vehicle(p.o., QD) RMC-6236(25 mg/kg, p.o., QD) RMC-6236(10 mg/kg, p.o., QD) AN9025(0.15 mg/kg, p.o., QD) AN9025(0.06 mg/kg, p.o., QD) 0 12 24 36 48 60 0 50 100 150 200 250 0 20 40 60 80 100 Time(h) Tumor Concentration (nmol/L) AN9025 Tumor PK/PD profile in HPAC(KRASG12D, PDAC) AN9025(0.2 mg/kg, p.o., QD*7) AN9025(0.1 mg/kg, p.o., QD*7) AN9025(0.04 mg/kg, p.o., QD*7) AN9025(0.2 mg/kg, p.o., QD*7) AN9025(0.1 mg/kg, p.o., QD*7) AN9025(0.04 mg/kg, p.o., QD*7) Tumor PD Tumor PK Tumor DUSP6 Level (% of vehicle) A B Figure 5. Pharmacokinetic (PK) and Pharmacodynamic (PD) profiles of AN9025 and RMC-6236 in HPAC xenograft models. AN9025 (A) and RMC-6236 (B) was administrated at indicated doses (p.o., QD*7). Tumor tissue and blood were collected 2, 4, 8, 24 and 48 hours post-dose to assess PD (DUSP6) and PK (blood concentration). 0 12 24 36 48 60 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 Time(h) Tumor Concentration (nmol/L) RMC-6236 Tumor PK/PD profile in HPAC(KRASG12D, PDAC) RMC-6236(25 mg/kg, p.o., QD*7) RMC-6236(10 mg/kg, p.o., QD*7) Tumor PD Tumor PK Tumor DUSP6 Level (% of vehicle) RMC-6236(25 mg/kg, p.o., QD*7) RMC-6236(10 mg/kg, p.o., QD*7) Background AN9025 forms a tri-complex with high affinity AN9025 inhibits the viability of RAS-addicted cancer cell lines AN9025 shows potent anti-tumor activity in vivo PK/PD correlation of AN9025 Conclusions AN9025 exhibits approximately 100-fold greater potency in inhibiting cell viability across RAS-mutant cell lines compared to RMC-6236. AN9025 displays a similar RAS-mutant sensitivity pattern to RMC-6236, with the hierarchy G12X>G13X≈Q61X>Wild Type. RAS mutations drive approximately 30% of human cancers, especially in pancreatic, lung, and colorectal cancers, representing a critical unmet clinical need. Pan-RAS(ON) inhibitors such as RMC-6236, bind to cyclophilin A (CypA) to form a binary complex which then engages the RAS (ON) protein to create a tri-complex that effectively inhibits downstream signaling pathways. RMC-6236 has shown encouraging clinical efficacy in Ras-mutated cancers. Here we report the preclinical characterization of a novel pan-RAS(ON) inhibitor, AN9025, with improved potency and a favorable PK/PD profile. !"#$%&'%()*+&,"-./0.1!(234)51 678.9:0 !"#$%&'()*'&$+*,-.!"+*!-!"#$%&'()*'&$+*,-.!"+*!- /012304502404.678#$"! /09510450530:.678#$"% 50:;40320340<.678#$"& 5054/0:20;5:0:.678#$"' 505;90120:5:0:.678#$(! 50;3:0320:550/.678)*$+ 50<:20420<5/0/.678,- 50<<<09504;0;=678,- 50<2/0250;30/>678,- =)?@*A@*BC@DDE&$?@*A@*B.678#$"!+FGG- AN9025 exhibits a 3- to 8-fold higher binding affinity for tri-complex formation and 1.5- to 2- fold greater maximum response compared to RMC-6236, suggesting enhanced formation of tri-complex by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able 2. Viability inhibition of RAS-addicted cancer cell lines by AN9025. IC50 values of AN9025 in RAS-addicted cancer cell lines harboring multiple RAS muta- tions or RAS WT were determined using the CellTiter-Glo (CTG) assay. Cells were treated with varying concentrations of the compounds for 72 hours. Figure 4. Anti-tumor activities of AN9025 in vivo. Tumor volume (left panel) and body weight change (right panel) during treatment with AN9025 at the indicated doses (p.o., QD) in HPAC, HepG2 and HCT116 xenograft models. The gray shading denotes the drug withdrawal period. Data represent mean±SEM; n=7-8 mice per group. ▪ We are actively seeking strategic partnerships with leading pharmaceutical and biotechnology companies to advance the development of AN9025 and facilitate its availability to patients in need. ▪ Contact information: alex.ye@adlainortye.com 0 100 200 300 400 Time(s) 0 0.5 1 Response(nm) KD2=8.8 nM Tri-complex formation induced by AN9025 GMPPNP-bound KRASG12D-AN9025-CypA A B 0 100 200 300 400 Time(s) 0 0.5 1 Response(nm) KD2=24.8 nM Tri-complex formation induced by RMC-6236 GMPPNP-bound KRASG12D-RMC-6236-CypA AN9025 exhibits more sustained DUSP6 inhibition compared to RMC-6236 across all tested doses. AN9025 achieved tumor regression in multiple CDX models harboring RAS mutation and demonstrated more sustained tumor suppression after drug withdrawal. The prolonged tumor DUSP6 suppression following AN9025 administra - tion suggests the potential for an intermittent dosing regimen to optimize tolerability and efficacy. AN9025 is a novel pan-RAS(ON) inhibitor developed by Adlai Nortye: Strongly binds cyclophilin A (CypA) with a slow dissociation rate, enabling tight tri-complex formation and potent RAS inhibition. Shows potent anti-proliferative activity in RAS-addicted cancer cell lines (picomolar IC50 values). Demonstrates a favorable PK/PD and tolerability profile in vivo. Induces deep tumor regression, with efficacy comparable to or ex- ceeding RMC-6236 in mouse CDX models. Is advancing through the IND-enabling stage AN9025 demonstrates potent inhibition of cell viability in RAS-addicted cancer cell lines at picomolar level.