引擎输出 · CRO RFQ/SOW

Request for Quote / Statement of Work — EGFR-C797S DCAF1 Degrader, First-Round Wet Validation

Sponsor: Catalyst AI (Quantum Longevity) · Program: brigatinib-class × DCAF1 EGFR-C797S PROTAC

Status: AI-prioritized, wet-ready degrader hypothesis — first-round go/kill validation.

Ask: quote for (A) custom synthesis of 3 bifunctional degraders + (B) a fixed cell-based degradation

go/kill panel. Bundled CRO (chemistry + biology) preferred; split bids welcome.

Objective: decide GO/KILL on the chemotype with the minimum spend. We are NOT asking the CRO to optimize — only to make 3 defined compounds and run 5 defined assays to defined thresholds.

A. Custom synthesis (3 compounds)

Bifunctional degraders = brigatinib-class EGFR warhead — linker — OICR-8268 (DCAF1 ligand). Target

10–25 mg each, ≥95% purity (HPLC), full QC (¹H NMR, LC-MS, HPLC purity), salt form chemist's

discretion. Single diastereomer at the OICR-8268 stereocenter (S, as drawn) preferred; racemic

acceptable for round 1 if it cuts cost/time (flag which).

ID	Name	Linker	MW	cLogP	RotB	Formula
CMC-1	brigatinib–PEG4–OICR8268	PEG4 (developability-optimized)	1212	9.2	29	C57H69Cl3F2N11O8P
CMC-2	brigatinib–piperazinyl–OICR8268	bis-propyl-piperazine (rigidified)	1176	9.5	22	C57H69Cl3F2N13O4P
CMC-3	brigatinib–C12-alkyl–OICR8268	C12 alkyl (potency control)	1176	13.0	27	C59H73Cl3F2N11O4P

Canonical SMILES:

• CMC-1: COc1cc(N2CCC(N3CCN(CCOCCOCCOCCOCCNC(=O)C[C@H](NC(=O)c4c[nH]nc4-c4ccc(Cl)cc4F)c4ccc(F)c(Cl)c4)CC3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2P(C)(C)=O)n1
• CMC-2: COc1cc(N2CCC(N3CCN(CCCN4CCN(CCCNC(=O)C[C@H](NC(=O)c5c[nH]nc5-c5ccc(Cl)cc5F)c5ccc(F)c(Cl)c5)CC4)CC3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2P(C)(C)=O)n1
• CMC-3: COc1cc(N2CCC(N3CCN(CCCCCCCCCCCCNC(=O)C[C@H](NC(=O)c4c[nH]nc4-c4ccc(Cl)cc4F)c4ccc(F)c(Cl)c4)CC3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2P(C)(C)=O)n1

Building-block notes (the CRO may already have these or quote them): brigatinib-class warhead = 2,4-

diaminopyrimidine + dimethylphosphine-oxide aniline, linker attached at the methylpiperazine/terminal

amine (the solvent-exposed tail); OICR-8268 = aryl-pyrazole-3-carboxamide DCAF1 ligand, linker attached

at the acetamide nitrogen. Both halves are literature compounds; synthesis is convergent

(warhead-linker fragment + amide coupling to OICR-8268).

B. Cell-based go/kill panel (5 experiments)

Run all 3 compounds (dose-response, ≥8 points, e.g. 1 nM–10 µM, technical triplicate) on:

1. Mutant-EGFR degradation (DC50 / Dmax). Isogenic engineered lines (Ba/F3 or PC9-derived)

expressing EGFR Del19/T790M/C797S and L858R/T790M/C797S. Readout: EGFR Western blot (vs

loading control / total-protein normalization) at 6 h and 24 h → DC50 and Dmax per compound/line.

2. WT-EGFR degradation → WT-sparing window. A WT-EGFR line (e.g. A431 or WT-EGFR Ba/F3) under the

same protocol → WT DC50/Dmax. Report the mutant-vs-WT degradation selectivity ratio (the decisive

safety number).

3. Pathway shutdown + viability. In the mutant lines: phospho-Western for pEGFR / pERK / pAKT

(±compound), and cell viability (CellTiter-Glo, 72 h) → confirm degradation tracks with signaling

shutdown and anti-proliferation, not non-specific cytotoxicity.

4. Mechanism confirmation (DCAF1-dependence). For the best degrader: **(a) DCAF1 (VPRBP)

knockdown/knockout rescue (siRNA or CRISPR) — degradation should be lost; (b) proteasome-inhibitor

rescue (MG132 or bortezomib) — degradation should be blocked; (c) EGFR ubiquitination** (IP-EGFR,

anti-Ub blot, ±compound). This proves the mechanism is DCAF1/UPS-mediated, not off-target.

5. Washout durability vs reversible TKI. Compare EGFR recovery after compound washout (e.g. 0/6/24 h)

vs a reversible 4th-gen TKI control at matched target coverage → demonstrate the durability advantage of

degradation over inhibition.

C. GO / KILL criteria (decision is the Sponsor's; these define a successful study)

• GO: mutant-EGFR DC50 < 100 nM (ideal < 30 nM) · Dmax > 70% · WT-sparing > 50× ·

pEGFR/pERK/pAKT shut down · DCAF1 knockdown rescues degradation.

• KILL: no degradation · WT-EGFR degraded as strongly as mutant · degradation only at cytotoxic

concentrations · DCAF1 knockdown does NOT rescue (mechanism is not DCAF1-mediated).

D. Deliverables

• CMC: compounds (≥10 mg, ≥95%), full analytical package (NMR/LC-MS/HPLC), CoA.
• Biology: per-compound DC50/Dmax tables (each mutant line + WT), WT-sparing ratios, phospho/viability

data, mechanism-rescue blots, washout curves — raw blots + quantified figures + a short summary.

• A single go/kill data table mapping each compound to the §C thresholds.

E. Logistics

• Timeline: please quote chemistry lead time + assay turnaround separately; target ≤ 12–16 weeks total.
• Quote: itemize (A synthesis per compound; B per assay/per line). Flag any compound whose synthesis

risk/cost is disproportionate (e.g. CMC-1 PEG4 vs CMC-3 alkyl).

• IP/confidentiality: compounds and results are Sponsor's; CDA required before disclosure of any

building-block detail beyond this brief. (Sponsor is conducting a separate formal FTO review;

CRO synthesis under CDA does not constitute an FTO clearance.)

• Reagents: isogenic mutant/WT-EGFR lines, DCAF1 KD/KO tools, and the reversible-TKI control — please

state which you have in-house vs need to source.

*This brief was generated from an AI-prioritized risk assessment. AI narrowed an open-ended EGFR-C797S

degrader problem to these 3 wet-testable molecules and 5 kill-or-go assays; the decisive endpoints —

DC50/Dmax, WT-sparing, DCAF1-dependence — are wet-only and are exactly what this study measures.*