Chemical genetics to chemicalgenomics: Utilizing small molecules

Suresh Venkateswaran: Proteomics andFunctional Genomics 412

Chemical Genetics

Study of biological systems using small molecule(chemical) intervention, instead of only geneticintervention. Such techniques useful for:

• Cell permeability and higher selectivity

• Perturbing protein function rapidly, reversibly, andconditionally

• Temporal and quantitative control

Forward/Reverse Genetics/Chemical Genetics

Challenges of Chemical Genomics

• Estimated 10x as many proteins thangenes in humans, since genes can codefor a multiple number of proteins.

• Most proteins have more than onefunction, hence a protein may require adifferent small molecule target for eachfunction.

Advantages of small molecules

• Most act reversibly. Difficult to construct conditionalalleles (ie. temperature sensitive mutations) or diminishpleiotropic effects.

• Conditional, quantitative, and temporal control, whereasgene knockout often produce steady-state effects ornone of the expected effects due to redundancy.

• Cell permeability and higher selectivity, unlike antisenseoligonucleotides and siRNA’s, which also neglect post-translational modification and individual proteindomain/sub-domain functions.

Disadvantages of small molecules

No general applications to discover cellular targets ormechanisms of any one small molecule!

• Selective small molecule ligands have highly specific proteinbinding partners

• Only a fraction of proteins have an identified ligand partner.

Must increase throughput of protein binding screens with acollection of small molecules to find viable protein-ligand pairsand correlate with phenotypic results to eventually makechemical genetics as generally applicable as genetics.

Searching for protein-ligand compounds

• Enzymes and receptors typically have smallmolecule partners that are easier to identify.

• Natural products (cyclosporine, rapamycin) aretypically highly selective and complex. However,most useful natural product come as mixturesthat must be purified. They are also so complexthat it is difficult to chemically synthesize (DOS:Diversity-Oriented Synthesis is an initiative toprovide detailed information on chemicallydiverse and structurally complex molecules)

Pre-selecting a target protein in proteinbinding screens

Small molecule target for Ure2p protein

Target idenitifcation strategies

Eliminating known targets or unwanted actions

• Screen small molecules inorganisms to inducefatality then prioritizethose “hits” with a varietyof mode of action assaysto eliminate unwantedmodes of action.

• For example, mostinhibitors of DHFR are notselective enough and areprobably toxic to mostmammalian cells

Applying Genetics

• Random mutations in a gene product to resisteffects of small molecules. Mutants arescreened with different small molecules and themutant that offers resistance is identified.

• A combination of non-lethal gene deletions canalso be identified that when present together canproduce an inviable phenotype, whereasindividual gene deletions lead to viable mutants(synthetic lethal screen).

Matching synthetic lethal profiles

Signaling proteins

• Protein kinases typically have well conservedactive sites. Mutating active sites creates greaterselectivity for small molecules.

• Temperature sensitive genetic studies Cdc28kinase suggested that primary function was tocontrol G1 to S phase transition in the cell cycle.Chemical genetics revealed that inhibitor-sensitive Cdc28 mutant arrested at G2/Mtransition.

Cytoskeleton

To induce mitotic arrest, monastrolwas synthesized, which led to thereorganization of the mitotic spindle.Monastrol inhibits an Eg5 kinesinprotein which diminishes microtubulemotility. The interaction is highlyreversible and specific, making thissmall molecule particularly useful forstudying Eg5.

Developmental Biology

Studying zebrafish embryos:

The 31N3 protein inhibits otolithdevelopment, which move inresponse to gravity and allowthe fish to maintain balance.31N3 allowed identification ofcritical time point of otolithdevelopment via temporal andquantitative control.

Sonic hedgehog pathwaySonic hedgehog pathway implicated in many developmentalprocesses including midline facial structures and limbs.Antagonists of the SHH pathway include:

Cyclopia

Stem Cells

Small moleculecan reverse thedifferentiatedstate of musclecells back intostem cells whichcan then be re-differentiatedinto differenttypes of cells

Protein-Protein Interactions• Enzyme/receptor proteins are generally the

easiest to modulate with small molecules.Challenges arise from the fact that:

– Proteins generally have more than one protein-binding surface.

– Proteins may lack small molecule binding sites sincethey are often flat and too large (800 Å2).

However, there are often “hot spots” on the binding surfacesthat account for most of the binding energy, which makeschemical genomics more practical for such applications

More examples• Serves as a molecular

antagonist of the oncogenic Tcell factor (Tcf/β-caretin proteincomplex (possible cancertherapy)

• Inhibition of LFA-1 and ICAM-1binding interaction haspotential to inhibit both theimmune and inflammatoryresponse.

Gene transcriptionSmall molecules canalso disrupt transcriptionand translation factors.For example, smallmolecules can be usedfor activating ribosomeactivity or changing RNAsecondary structure.These domains areknown as “riboswitches”

Coenzyme B12

ConclusionsChemical genetics is useful for many properties,including cell permeability, high selectivity, rapidperturbation of protein function, reversibility, andcondition temporal and quantitative control. Thesetechniques, when combined with genetic tools, willyield a more complete understanding of biologicalprocesses, diseases, and future drug treatments.More studies and higher throughput is required tobefore chemical genetics is as applicable andgeneralizable as modern day genetics.