In this work, we developed a computational workflow to mine the Protein Data Bank for isosteric replacements that exist in different binding site environments but have not necessarily been identified and exploited in compound design. Taking phosphate groups as examples, the workflow was used to construct 157 data sets, each composed of a reference protein complexed with AMP, ADP, ATP, or pyrophosphate as well other ligands. Phosphate binding sites appear to have a high hydration content and large size, resulting in U-shaped bioactive conformations recurrently found across unrelated protein families. A total of 16 413 replacements were extracted, filtered for a significant structural overlap on phosphate groups, and sorted according to their SMILES codes, see workflow in Figure 1. In addition to classical isosteres of phosphate, we found unexpected types of replacements that do not conserve charge or polarity, for example phosphate replaced by aliphatic groups, phenyl, or carbamoyl groups. The structural mechanism involved in structural isosteres appears varied: New interactions may be created, water molecules are important, in some case ion plays a role, and of course large and small conformational changes do occur at the binding sites. This study has implications both in the field of medicinal chemistry, i.e. it expands our knowledge of structural isosteres, and in the field of chemoinformatics, since our results have implications with respect to the definitions of chemical similarity.

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Structural isosteres of phosphate groups
in the protein data bank
Alexandre Borrel, PhD
Postdoctoral Research Associate,
Department of Chemistry, Bioinformatics Research Center,
North Carolina State University, USA
Division of pharmaceutical chemistry and technology
Faculty of pharmacy
University of Helsinki
@AlBorrel

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Background: Drug optimization
Hann, M.M. (2011). Medchemcomm 2: 349–355.
Modify a drug to influence
Absorption, Distribution,
Metabolism, Excretion and
Toxicity (ADME-Tox) properties
in preserving an initial
biological activity.Initial hit
Final drug

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Definitions
(1) Brown, N. (2014). Mol. Inform. 33: 458–462.
Isostere or isosteric replacement (1):
• Preserve biological activity
• Retain the physicochemical or topological properties of the reference compound
Carcinogen Analgesic
Anti-inflammatory
Cheminformatics to propose possible replacement

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In silico approaches
Propose large databases of bioisosters, ligand based and structure based
sc-PDB-frag (1):
- Structure based
- Interaction fingerprints
KRIPO (2):
- Structure based
- Pharmacophore
fingerprints
SwissBioisostere (3):
- Ligand based
- Molecular pair from
CHEMBL
Hydrophobic
H-bond
(1) Desaphy J, Rognan D (2014) J Chem Inf Model 54:1908–1918. doi: 10.1021/ci500282c
(2) Wood DJ, Vlieg J De, Wagener M, Ritschel T (2012) J Chem Inf Model 52:2031–2043. doi: 10.1021/ci3000776
(3) Wirth M, Zoete V, Michielin O, Sauer WHB (2013). Nucleic Acids Res 41:1137–1143. doi: 10.1093/nar/gks1059

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Local structural replacements
Chemical groups which occupy the same space
in two homologous superimposed complexes.
Local structural
replacement (LSR)
Superimposed
homologous proteins

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Study case: phosphate
• Attractive target for therapeutic development (1)
• 30% of the cellular proteins are phosphoproteins,
2,940 of structures available in PDB
• Phosphate group is charged at biological pH, poorly
permeable (2)
Phosphate groups in ATP
(1) Cohen, P. (2000). Trends Biochem. Sci. 25: 596–601.
(2) Smith, F.W., Mudge, S.R., Rae, A.L., and Glassop, D. (2003). Plant Soil 248: 71–83.

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Computational workflow
Extract from PDB (130,000 structures) complexed
protein which included ligand with a phosphate
group

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Computational workflow
Filter the PDB
(1,186 structures)

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Computational workflow
Homologous protein
(Blastp cutoff 10-100)
10,991 structures

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Computational workflow
LSR

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Computational workflow
15,819 phosphate replacements

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Hierarchical organization
Replacement containing (cycle, P, B, ….)

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Hierarchical organization
Replacement containing (cycle, P, B, ….)
• 16 Protein family, PDB information (KS =
Kinase)
• 70 clusters (30% of identity sequences)
KS-5_4I3Z
(Kinase – cluster 5 – PDB ID: 4I3Z)
Replacement containing (cycle, P, B, ….)

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Hierarchical organization
Replacement containing (cycle, P, B, ….)
Replacement containing (cycle, P, B, ….)
• LGD (Ligand)
• LSR (Local Structural
Replacements)
• BS (Binding site, d = 4.5 Å)
• 16 Protein family, PDB information (KS =
Kinase)
• 70 clusters (30% of identity sequences)

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Clustering navigation

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Isosteres?
Number of atom similar - different
LigandID–PDBIDIC50
Extract affinity information (from congener series)

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U-shape configuration
U-shape replacements, found in different protein families (binding affinity decreases).

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Miscellaneous replacements
Hydrophobic and positively charged replacements open perspectives to find not
suspected before.
Hydrophobic replacements
NAD+ truncated (positive)

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Conclusion
• 15,819 phosphate replacements
• Organization based on target and type of structural replacements
• Mechanisms for phosphate replacements (protein or metal displacement)
• New no suspected replacement (U-shape, hydrophobic, or positively charged)
• Workflow is fully customizable (github)
Zhang, Y.*, Borrel, A.*, Ghemtio, L., Regad, L., Boije af Gennäs, G.,
Camproux, A.-C., et al. (2017). Structural Isosteres of Phosphate Groups in
the Protein Data Bank. J. Chem. Inf. Model. 57: 499–516.

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Acknowledgments
Authors:
- Yuezhou Zhang
- Leo Ghemtio
- Leslie Regad
- Gustav Boije af Gennäs
- Anne-Claude Camproux
- Jari Yli-Kauhaluoma
- Henri Xhaard*
Fourches’ lab

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Water molecules versus
RX
• Poorly crystallized
• Present only at very high resolution
(< 1.5 Å)
Datasets from the PDB with 1.5 Å and 3 Å of
resolution

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Miscellaneous
replacements
Hydrophobic replacements, favour hydrophobic contacts in
binding site.
Positively charged replacement is surprising
considering that the phosphate groups are
negatively charged.

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Bioisoster replacement
“One of two or more substances related to each other by origin, structure, or
function.” (IUPAC)
Shin, Y., Chen, W., Habel, J., Duckett, D., Ling, Y.Y., Koenig, M., et al. (2009). Bioorganic Med. Chem. Lett. 19: 3344–3347.
A group change and the
affinity (IC50)

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Hierarchical organization
Dependency of protein target: LSR target based classification
Meanwell, N.A.N.N. a (2011). J. Med. Chem. 54: 2529–2591.
cPLAA2α inhibitor analogs
+ + + - - -
Angiotensin II receptor antagonist analogs

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Structural replacement
ESP: electrostatic
score potential (1)
= 1: phosphate is
fully covert by the
LSR
LSR superimposition not overlap completely with phosphate groups

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Replacement
mechanisms
PDB code: 3JZI – 1DV2

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Replacement
mechanisms
PDB code: 3JZI – 1DV2

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Replacement mechanisms
PDB code: 3JZI – 1DV2
Protein replaces the phosphate group

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Chemical replacement
Southall, N.T., and Ajay (2006). J. Med. Chem. 49: 2103–2109
Example of chemical replacements
in kinase patent space from gefitinib
remove
Replaceadd