## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: Ki
- Label type: continuous
- Binary semantics: pchembl
- Assay description: HPK1 Biochemical Enzyme Assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay (MSA). The reactions were conducted in 50 μL volumes in 96-well plates, and contained 0.5 nM human full-length recombinant HPK1, 3 μM phosphoacceptor peptide, 5FAM-AKRRRLSSLRA-COOH (CPC Scientific, Sunnyvale, Calif.), test compound (11-dose 3-fold serial dilutions, 2% DMSO final) or DMSO only, 0% Tween-20, 1 mM DTT and 2.5 mM MgCl2 in 50 mM MOPS (3-(N-morpholino)propanesulfonic acid), pH 7.8, buffer and were initiated by addition of 75 μM ATP, following a 20-min preincubation. The reactions were conducted for 60 min at 37° C., stopped by the addition of 50 μL of 0.01 M EDTA, pH 8, and the extent of reactions (15-20% conversion with no inhibitor) was determined after electrophoretic separation of the fluorescently labeled peptide substrate and phosphorylated product on an LabChip EZ Reader II (PerkinElmer, Inc., Waltham, Mass.).Inhibition of HPK1 was also measured using the fluorescence based chelation-enhanced fluorescence (CHEF) method (1), using a proprietary fluorescent peptide substrate, in which a cysteine residue is alkylated with a sulfonamido-oxine based derivative to afford an amino acid termed C-Sox (CSx). The assay was conducted similarly as described for the MSA method above, but using 3 μM Ac-[CSx]HSLPRFNR-amide peptide substrate (also known as AQT0178 when purchased from AssayQuant Technologies Inc., Hopkinton, Mass.) and 45 μM ATP. Initial reaction velocities were determined by following the peptide fluorescence (λex=360 nm, λem=500 nm) at 30° C. for 15 min in a Tecan M1000 plate reader (Tecan Group Ltd., Männedorf, Zürich, Switzerland). The inhibition constant (Ki) values were calculated by fitting the % conversion based (MSA method) or fluorescence based initial velocities (CHEF method) to the Morrison equation (2) for tight-binding competitive inhibition using non-linear regression method and an experimentally measured ATP Km (29 μM by MSA and 19 μM by CHEF, respectively). The inhibitors were shown to be ATP-competitive from kinetic and crystallographic studies. HPK1 protein was produced in-house and preactivated by autophosphorylation of enzyme with MgATP as described in the section Production of recombinant autophosphorylated full-length HPK1.
- Assay organism: Homo sapiens
- Target name: Mitogen-activated protein kinase kinase kinase kinase 1
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: CCn1c(C)nnc1-c1cccc(N2Cc3c(cc(C4(C)CC4)nc3CN)C2=O)n1
- Properties:
- ChEMBL ID: CHEMBL5942272
- Molecular weight: 403.49
- Heavy atoms: 30
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 6
- TPSA: 102.82
- LogP: 2.73
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.41
- QED: 0.7

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 8.82
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type Ki; relation =; value 1.52; units nM; pChEMBL 8.82; comment 1097946

## Query Molecule B
- Canonical SMILES: CCn1c(C)nnc1-c1cccc(N2Cc3c(cc(N4CCCC4(C)C)nc3CN)C2=O)n1
- Properties:
- ChEMBL ID: CHEMBL5936769
- Molecular weight: 446.56
- Heavy atoms: 33
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 7
- TPSA: 106.06
- LogP: 3.06
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.46
- QED: 0.64

## Molecule Similarity
- Weighted Tanimoto: 0.73

Answer: 9.82