Kalliokoski T, Olsson TSG, Vulpetti A. J. Chem. Inf. Model. 2013, 53, 131-141.

SubCav - Tool for subpocket comparison and alignment Dr. Tuomo Kalliokoski Lead Discovery Center GmbH, Dortmund, Germany

Work conducted at Novartis Institutes for Biomedical Research, Basel, Switzerland

Protein Databank (PDB) is growing

• 100000 Number of searchable structures 1972-Mar 2013 90000 80000 70000 60000 50000 40000 30000 20000 10000 0

How many fragments are there?

8 million unique chemical structures 2 million lead-like structures 400,000 Rule-Of-Three compliant structures Zuegg and Cooper. Drug-Likeness and Increased Hydrophobicity of Commercially Available Compound Libraries for Drug Screening. Curr Top Med Chem 2012, 12, 1500-1513.

Bridging “Structural”-Space and “Fragment”-Space

The information content of PDB is increasing Fragment chemical space is too large for experimental Fragment-Based Drug Design (FBDD) The need to develop tools for FBDD to take advantage of PDB!

Binding site similarity

“The availability of such data provides a basis for the identification of bioisosteres that are target specific. The resulting bioisosteres might be expected to provide more reliable information when modifying an existing lead compound than do existing approaches, which are based either on empirical measures of inter-substituent similarity or on non-target specific crystallographic data.” Kennewell EA, Willett P, Ducrot P, Luttmann C. Identification of target-specific bioisosteric fragments from ligand–protein crystallographic data. J Comput Aided Mol Des 2006, 20, 385-394.

Subpockets and fragments

BRICS*

*Degen J, Wegscheid-Gerlach C, Zaliani A, Rarey M. On the art of compiling and using ’drug-like’ chemical fragment spaces. ChemMedChem 2008, 3, 1503–1507.

SubCav

• • • Tool for subpocket similarity searching and alignment Based on pharmacophoric fingerprints with geometric hashing-inspired alignment Source code available via [email protected]

SubCav atom type Acceptors with sp2 character (π acceptor) (A=) α-carbon (CA) Donor (D) Donors with sp2 character (π-donor) (D=) Hydrophobe (H) π-hydrophobe (H=) neutral donor & acceptor (P) Ignored

Fingerprint descriptor

PDB atom types

ALA.O ARG.O ASN.O ASN.OD1 ASP.O ASP.OD1 ASP.OD2 CYS.O GLN.O GLN.OE1 GLU.O GLU.OE1 GLU.OE2 GLY.O HIS.O ILE.O LEU.O LYS.O MET.O PHE.O PRO.O SER.O THR.O TRP.O TYR.O VAL.O

ALA.CA ARG.CA ASN.CA ASP.CA CYS.CA GLN.CA GLU.CA GLY.CA HIS.CA ILE.CA LEU.CA LYS.CA MET.CA PHE.CA PRO.CA SER.CA THR.CA TRP.CA TYR.CA VAL.CA

LYS.NZ

ALA.N ARG.N ARG.NE ARG.NH1 ARG.NH2 ASN.N ASN.ND2 ASP.N CYS.N GLN.N GLN.NE2 GLU.N GLY.N HIS.N ILE.N LEU.N LYS.N MET.N PHE.N SER.N THR.N TRP.N TRP.NE1 TYR.N VAL.N

ALA.CB ARG.CB ARG.CD ARG.CG ASN.CB ASP.CB CYS.CB CYS.SG GLN.CB GLN.CG GLU.CB GLU.CG HIS.CB HIS.CG ILE.CB ILE.CD1 ILE.CG1 ILE.CG2 LEU.CB LEU.CD1 LEU.CD2 LEU.CG LYS.CB LYS.CD LYS.CE LYS.CG MET.CB MET.CE MET.CG MET.SD PHE.CB PRO.CB PRO.CD PRO.CG SER.CB THR.CB THR.CG2 TRP.CB TYR.CB VAL.CB VAL.CG1 VAL.CG2

HIS.CD2 HIS.CE1 PHE.CD1 PHE.CD2 PHE.CE1 PHE.CE2 PHE.CG PHE.CZ TRP.CD1 TRP.CD2 TRP.CE2 TRP.CE3 TRP.CG TRP.CH2 TRP.CZ2 TRP.CZ3 TYR.CD1 TYR.CD2 TYR.CE1 TYR.CE2 TYR.CG TYR.CZ

HIS.ND1 HIS.NE2 SER.OG THR.OG1 TYR.OH

PRO.N and all HETATM D=

Bin Range ( Å)

1 2.1-4.5

2 4.5-6.3

3 4 6.3-8.0

8.0-10.0

9.3Å= 4 3.4Å= 1 A= CA 6.0Å= 2

Alignment algorithm

Implementation details

Validation study

• • • Align pairwise all similar subpockets in PSMDB* (non-redundant subset of PDB) 3,268,620 pairs from 3,886 PDBs with 17,044 subpockets with 332 different fragments Two alignment methods: – Fragment-based alignment – SubCav-based alignment * Wallach I, Lilien R. The Protein–Small-Molecule Database (PSMDB), A Non-Redundant Structural Resource for the Analysis of Protein-Ligand Binding, Bioinformatics 2009, 25, 615-620.

•

When are two subpockets similar?

Two subpockets are similar if both after alignment have – Root-Median-Square-Deviation (RMSD) of fragments found in subpockets is less than 1.5 Å – Enough matched features*

RMSD = 1.00

Overlap = 0.79

*Matched feature=if two features from the two subpockets are within 1 Å distance

Very rarely subpockets with same fragments are geometrically similar...

3500000 3000000 2500000 2000000 1500000 1000000 Fragment-based OK SubCav- based OK Both OK Not matched 500000 0 0,5 0,6 0,7 0,8 0,9 1

120000 SubCav finds 73%-85% of fragment based (plus something else!) 100000 80000 60000 40000 20000 Fragment-based OK SubCav- based OK Both OK 0 0,5 0,6 0,7 0,8 0,9 1

Three structures of thrombin aligned. The query (magenta) fragment-aligned (green) vs. SubCav aligned (cyan)

Bioisosteric replacement example

ACP

Heat Shock Protein 90 (HSP 90)

Bioisosteric replacement example

Escherichia coli DNA gyrase B (sequence similarity 30%)

Bioisosteric replacement example

Adenine -> pyrazole?

Escherichia coli DNA gyrase B (sequence similarity 30%)

Bioisosteric replacement example

HSP90 inhibitor

Analysis of Histone Methyl-Transferase Binding Sites S-adenosylmethionine (SAM) or S-adenosyl-l-homocysteine (SAH) Fragmented in three: adenine, ribose, and tail fragments

Pairwise SubCav-alignment and hierarchical clustering based on Overlap

Analysis of Histone Methyl-Transferase Binding Sites

The clustering of the cofactor binding site by subpockets around each specific fragment revealed different levels of local similarity within the selected proteins set.

Analysis of Histone Methyl-Transferase Binding Sites

The clustering of the cofactor binding site by subpockets around each specific fragment revealed different levels of local similarity within the selected proteins set.

Analysis of Histone Methyl-Transferase Binding Sites A B C D

Take home message

Subpocket analysis can provide ideas in CADD

Acknowledgements

• • • Novartis Institutes for Biomedical Research: – Dr. Anna Vulpetti (mentor & co-author) – Education office (Presidential Postdoctoral Fellowship) Cambridge Crystallographic Data Centre: – Dr. Tjelvar Olsson (mentor & co-author) Chemical Computing Group: – Dr. Guido Kirsten (idea for alignment protocol)