Can field based chemistry help us to predict protein-DNA binding sites?
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Can Field-Based Chemistry Help Us To Predict Protein-DNA Binding Sites? Daniel Barr, PhD Assistant Professor of Chemistry Utica College [email protected]
• The ABC’s of DNA – Direct vs. Indirect Readout– Field-based approach to understanding DNA– Electrostatics as a “shortcut” to dynamics?
• Implications for Pharmaceuticals– Design small(-ish?) molecules to bind DNA– Utilize a variety of peptidomimetic backbones– Grow fragments to hit flexible/dynamic sites
Sequence-Specific Protein-DNA Binding• Direct readout recognizes the patterns of
hydrogen bond donors/acceptors unique to each DNA sequence
• Indirect readout recognizes the shape and/or dynamical flexibility of the DNA sequence– Can be sequence-dependent
Rohs et. al. Ann.Rev.Biochem. 2010
Presenter
Presentation Notes
Each of the 4 possible base pairs is distinguishable in the major groove, in the minor groove can only distinguish AT or GC A-tracts tend to bend into the minor groove; long GC tracts tend to bend into the major groove
Direct Readout
Suzuki, Structure 1994
Sequence-Dependent Flexibility
• In general YR tends to be most flexible
• AT basepairs less context-dependent than CG
Packer, Dauncey, HunterJMB 2000Lavery et al (ABC Consortium)Nucl Acid Res 2009
YRRY
RRSpiriti et al (in preparation)
Protein-DNA Contacts• Q18, R22 (blue)
are bidentate ligands: direct readout
• Y7, Y17 (red) are monodentate: indirect readout
Barr and van der Vaart, PCCP 2012
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Red=residues which increase in contact to DNA as bending angle increases Blue=residues which decrease in contact to DNA as bending angle increases
Analysis of DNA with Forge
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Cresset software gives us the best chance to understand the DNA without full dynamics studies Try to compare protein and DNA fields to see if we can gain insight into specificity Big files are causing problems – need to load each strand as a separate molecule which makes it difficult to align.
Neighbor Sequence Comparisons
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Top Left: AAAC, Bottom Left: AAAG, Top Right: AAAA, Bottom Right: AAAT Observations: 1. It’s tough to get the rotations exactly correct. 2. There are lots of similarities, but the differences in the 4th basepair are apparent and have a noticeable impact on the third basepair. AAAC and AAAA are the most different (0.947 similarity in Forge) – change bp and change the order of purine/pyrimidine. AAAT is less similar (0.968) than AAAG (0.973) – y/r seems to matter more than bp step.
Inverted Sequence ComparisonsCAAA AAAC
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Presentation Notes
Left: AAAC, Right: CAAA NOT inverse-symmetric! This is important – context
Drug Design Strategy• DNA-binding drugs may need to be large-ish
– It might not be sufficient to hit one or two key sites– Might need bigger molecules with specific
geometries to account for DNA bending
• Test different scaffolds as templates for peptidomimetic backbones
The point of all of this is that the peptide’s amide bonds will be easily hydrolyzed by biological enzymes The synthetic amides are different enough to be stable (don’t follow the same backbone scaffold, don’t have recognizable side chains, etc).
Drug Design Step 1• Use Forge to align models against the target peptide
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Presentation Notes
If our clever creations didn’t work well, it’s some solace that the “standard” mimetics don’t work very well either. Note particularly the difficulty in positioning the arginines…
Drug Design Step 1• Use Forge to align models against the target peptide
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Problems: Not clear how to build the side chains – the structure is different enough from the peptide to make this a non-trivial task. What degree of flexibility needs to be incorporated? Alignments are VERY low – (a) overwhelmed by backbone charges, (b) can’t decide where the side chains belong (3 and 4 are “correct”).
Drug Design Step 1• Use Forge to align models against the target peptide
Presenter
Presentation Notes
Removing the backbone carbonyls helped – the scores increased noticeably, but still not enough. Similarity is still only ~50% and the arginine is pointing the wrong direction on some alignments May need to get rid of the nitrogens in the reference structure as well as the carbonyls?
Drug Design Step 2• Use Spark to grow candidates to the target peptide
Presenter
Presentation Notes
Problems: Not clear how to build the side chains – the structure is different enough from the peptide to make this a non-trivial task. What degree of flexibility needs to be incorporated? Alignments are VERY low – (a) overwhelmed by backbone charges, (b) can’t decide where the side chains belong (3 and 4 are “correct”).
Ongoing/Future Work
• Extend the DNA sequence analysis to all 39 nearest-neighbor combinations
• Keep working on fragment growing for drug candidates
• Docking studies?
Acknowledgements
• Heather McManus and Daniel Bollen (DNA sequence comparisons)• Michael Convertino and Jade Bonsel (drug scaffold analyses)• Gabrielle Abbot (drug design and fragment growing)
– Cresset Bio-Medical Discovery– National Science Foundation – Utica College
