Targeted Protein Degradation at Syngene - AWS

Targeted Protein Degradation at Syngene

2 Targeted Protein Degradation at Syngene

Syngene has extensive experience in PROTACs

We have a team of 130 FTEs working on the synthesis of PROTACS for various clients

We have synthesized full degrader molecules from milligram to gram quantity

We have extensive experience working on modification of CRBN and VHL ligands, multiple spacers and linkers

We have experience of running the primary screening assays and DMPK studies for this class of molecules.

We were able to help our partners identify multiple lead optimized compounds through optimization of the core, linker and the CRBN ligands and one of them is now being processed through tox studies.

The team has considerable experience solving the synthetic chemistry and purification challenges with PROTACS.

Experienced in synthesis of linkers and scaffold in multigram quantities.

3 Targeted Protein Degradation at Syngene

PROTACs expertise – Single compound mode

Small molecule inhibitor

CRBN

POI

Successfully optimized the different regions:

• Small molecule inhibitor, novel IP generation

• Spacer: length and orientation

• Linker: length

• CRBN ligand

Achieved pico -molar potency with excellent metabolic stability and PK profile

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PROTACs expertise – Targeted library synthesis

• >90 libraries completed with >99 % success rate; LCMS purity > 90 % and each library consisted of 24 compounds

• All compounds processed through high throughput MS directed purification

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PROTAC – Degrader optimization expertise: CRBN ligand

Small molecule inhibitor

POI

Small molecule inhibitor

Small molecule inhibitor

Small molecule inhibitor

POI

Small molecule inhibitor

POI

Small molecule inhibitor

• Purification of degraders through prep. HPLC, C18 columns and silica gel chromatography

• Chiral separation of CRBN ligands through SFC

• Expertise in synthesizing linker acids and amines with E3 ligases in multigram quantities

• PROTAC degraders are synthesized on a scale of 5 mg to 5 g for advanced studies (PK/PD)

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PROTAC – Degrader optimization expertise: VHL ligand

• Developed process for the synthesis of VHL1 and prepared at 700 g scale• Improved from overall yield of 32% in 7 steps to 58% in 5 steps • Avoided column purification in all the steps except 2nd

• Developed process for synthesis of VHL-2 and 3 and prepared more than 100g ligand following 8 to 10 linear synthetic steps• Delivered 15 g of each linker attached VHL-ligand with synthesis of linker, core and their coupling (amide or ether bond

reactions) • Various linker preparations achieved (C linkers & PEG linkers) in multigram scale with varying linker size (3 to 16 carbon for C

linker & 3 to 6 PEG linker) and different functional groups (-NH2, -Cl, -COOH).

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From library synthesis to lead compounds: A case study

Targeting Ligand variations Spacer

variations

Linkervariations

Total number of scaffolds synthesized: 46

Average number of steps for the scaffold synthesis: 6-7

Total number of library reactions: Approximately 1100

Time taken to move to lead optimization: 11 months (scaffold synthesis followed by library Synthesis)

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PROTAC-based screening platforms at Syngene

Cell-free proximity assay using AlphaScreen

(Ternary complex dimerization assay)

CRBN: Target ProteinProtein: Protein Interaction Assay

• Protein dimerization, binding (FP assays)

• HiBit Assays, ADP-Glo (kinase activity)

• Solubility, viability assays• Phospho assays

HiBiT Lytic Cell Based Functional Assay

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In Vitro ADME and PK profiling to support PROTACs targets

In vitro ADME Assays

• Thermodynamic solubility in simulated intestinal fluids • LogD (LC-MS/MS based): Ability to measure LogD values upto 6.0 units • Plasma and whole blood stability • Metabolic stability in hepatocytes (mouse –most preferred, rat, rabbit, dog, monkey and human)• Bidirectional Caco-2 permeability/LLC-PK1-MDR1- Specially designed for PROTACS• UC-PPB (custom based with stability assessment) and RED• Cocktail CYP inhibition• Time dependent CYP inhibition (need based)• Metabolite identification (value added services) in hepatocytes- specific to PROTACs• Preclinical formulation development and stability studies

PK studies

• Cassette PK studies in mice• Single dose oral and IV PK studies• Tissue exposure studies• Blood and plasma PK studies

For more information, contact [email protected]

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