1. Function first: a powerful approach to post-genomic drug discovery Stephen F. Betz, Susan M. Baxter and Jacquelyn S. Fetrow GeneFormatics Presented by Jamie Duke April 7 th, 2004

2. 2 Goal: To answer this question: – How can we effectively use the new information from the genome sequencing projects to accelerate the development of new therapeutics that target gene products or their functions?

3. 3 A “deluge of targets” There is an overwhelming amount of nucleotide sequences generate from high- throughput sequencing and differential expression methods Targets are not necessarily even expressed in vivo The actual targets are waiting to be discovered

4. 4 Post-genomic drug discovery Methods must be able to deal with a large number of targets General strategy relies on high-throughput sequencing of large compound libraries against target proteins – Requires a knowledge of enzymatic activity, or binding against a known ligand Methods are costly, number of targets analyzed are limited, and a second assay is generally required

5. 5 Screening Strategies Use of nuclear magnetic resonance (NMR) and x-ray crystallography Structure based drug design has also come into play with therapeutics Both strategies still require analyzing single proteins serially – Best method for the future involves automation

6. 6 Screening Strategies Experimental efforts are resource intensive, and limited to proteins that can be cloned X-ray Crystallography is only possible if the protein can form diffraction-quality crystals NMR is only possible if the protein is well behaved in solution Structural biology is only possible with high quality structures

7. 7 Structure Structural prediction is neither easy nor cheap Knowing tertiary structure does not guarantee the transfer of function or small molecule binding sites Inference of function from similar sequences with known function is correct less than 50% of the time – A “similar sequence” is a sequence that is 30% or more identical, most proteins do not meet this requirement – Additionally, different structures have been known to support the same activity

8. 8 Selectivity The aim is to develop truly selective compounds from the beginning of the discovery process Decrease the failure rate of compounds in development and ultimately lower cost and time

9. 9 Function in Drug Discovery Drug discovery starts by determining the function of the drug leads from mining the genomic data – Pathway involvement, catalytic activity, protein class or active-site chemistry Functional features can be used to develop assays for a more straightforward path “Parallel large-scale processes and analyses to identify function first will be key for this lead discovery approach to be successful in the post- genomic era.”

10. 10 Function Assignment Function of the sequences is often inferred through sequence “similarity” – Function is automatically transferred, and can lead to misannotation and misinterpretation SAGE and parallel protein analysis are generally used – These experimental procedures allow for the gene product function to be identified in a complex environment yielding data which is used to validate the target – Unfortunately, low copy genes and a high false positive rate limit the use of these methods

11. 11 Function first approach to structural and chemo-proteomics Process starts with the identifying a set of protein sequences in the human proteome – Looking for sequence that have particular binding sites, carries out catalysis, or has been previously identified Proteins are classified by their functional sites – Analysis of the families is key to specific drug design Structures of family members are determined using protein folding algorithms Small molecule binding sites are identified This approach saves crucial time and money in the drug discovery process

12. 12 Approximate Structure Analysis For each protein, an approximate model is generated Algorithms developed by Jeffrey Skolnick for the CASP competitions are used to predict the structure Models are not perfect due to imperfect scoring functions and energy potentials

13. 13 Fuzzy Functional Forms™ A technique to identify biochemical function An FFF is a motif that describes the chemistry and geometry of the functional site within the protein Information is based upon known structures in the PDB – Functional residues are identified in related protein structures – Residues are selected based on the nature of the function, chemistry or structure of the site – Geometric constraints are defined for key residues in the structure

14. 14 Functional Family Approach Functional sites are (generally) well conserved in families FFF’s are used to determine all proteins in the genome with the given functional site Functional families are identified by: – Sites identified by FFF, and – Computational information on the functional site that yields valuable biologically relevant data necessary for drug discovery

15. 15 Functional Family Approach The ultimate goal is to identify small molecules that will selectively inhibit a single member of a family, thus reducing interaction with other proteins This approach allows for the account and classification of functionally related proteins Provides a better assurance of the “druggability” for a putative target

16. 16 Complementary to Cell Based Information This method allows for efficient target validation due to parallel identification Allows for large-scale identification of function and structure without large-scale investments Provides information that is relevant to assays being run to determine functionality and interpretation of microarray data

17. 17 Rapid Analysis of the Structure Because the structure of the protein does not need to be determined to the atomic level, it is much less computationally intense Far fewer protein folding is done in silico because only models identified through FFF or high scoring models are folded The process is automated – Scientists can compute more than 25,000 protein sequences and make structure-function assignments in weeks, not months or years as would take to serially test each sequence through the experimental techniques The combination of structure and function information that allows for more reliable assignments than sequence based methods

18. 18 Alternative Drug Binding Sites One key feature of FFF is it’s ability to identify multiple active sites for one protein A protein may be annotated as a phosphatase, but it may also have a catalytic site, a metal binding site, and a regulatory site, as does serine-threonine phosphatase Alternative sites are also potentially druggable The information from multifunctional

19. 19 Binding Sites

20. 20 Key Information It is key to know the structure of the functional site – Recognition of the similarities and differences among a set of potential targets allows for designing specific small molecules that are specific for each member of the family

21. 21 Conclusion The function first approach provides an effective way to mine the genomic data to lead to compounds that can be developed into drugs Using this method, in association with biological, structural, and chemical methods will lead to drug discovery that is more efficient, and cost effective