Deletion library construction

Gene overexpression Haploinsufficiency Deletion

Complete deletionSingle deletionMulticopy plasmid

Antibiotic resistance gene

Unique ID sequences

Control

SM DNA

PCR Cy5

DNA

CombineEnriched

Depleted

DNA barcode arraySurvivors

Constant

With SM, strain was...

PCR Cy3

Wildtype

Figure 2.5 Typical SM-induced growth alteration screening procedure.

m/z

Time

Detection

Figure 2.7 FAC-MS: SM affinity is proportional to retention time.

Proteinapplication

e−

Fe(CNFe(Fe(CNCN)63−

Fe(CN)63−

Figure 2.9 Typical EIS device. Auxiliary, working, and reference electrodes are shownin gold, blue, and green, respectively. In a solution (gray transparent cube), a redox com-pound such as Fe(CN)6

3− will “complete the circuit” between the auxiliary and workingelectrodes, but upon application of a protein that would bind to a SM immobilized onthe working electrode, the access of the redox solute may be severely limited, resultingin impedance changes.

(a)

Styrene GMA DVB

Emulgen1150S -60

SeedPolymerizationPolymerization

GMA

MP

Compound X

OO

50

H

MP-DL

(b)

DMF THF Ethyl Acetate

Dioxane Toluene Dichloromethane

Water

(c)

268.9 ± 44.4228.9 ± 45.6225.5 ± 39.5From O to W

153.5 ± 15.4182.0 ± 40.9219.8 ± 31.3From W to O

DichloromethaneTolueneDioxane

222.5 ± 36.3210.4 ± 38.4223.8 ± 21.0From O to W

250.6 ± 31.3220.3 ± 30.5222.4 ± 35.8From W to O

Ethyl acetateTHFDMF(d)

Figure 3.5 (a) Synthetic scheme of FG beads; (b) FE-TEM image of the isolated FGbeads; (c) photo image of FGNE beads dispersed in DMF, THF, ethyl acetate, 1,4-dioxane,toluene, and dichloromethane (these dispersions contain 0.4 mg of FGNE beads); (d)dynamic light-scattering (DLS) analyses of FGNE beads in DMF, THF, ethyl acetate, 1,4-dioxane, toluene, and dichloromethane and water-resuspended beads from each organicsolvent.

Streptavidin-coated well

Biotinylated small moleculeimmobilization on the

streptavidin-coated well

Phage affinity selection usingcloned phage cDNA libraryagainst biotinylated small

molecule

Amplified phage clone sequencing& target identification

Affinity-based bound phageamplification

Repeated Phage Panning

Figure 5.1 Overall phage display biopanning scheme. Identification of a target proteinof a small molecule.

a: DMSO (5%) control

b: HBC 3.125 µM

c: HBC 6.25 µM

d: HBC 12.5 µM

e: HBC 25 µM

f: HBC 50 µM

g: HBC 100 µM

ka (1/Ms): 367kd (1/s): 2.98 x 10−3

KA (1/M): 1.23 x 105

KD (M): 8.11 x 10−6

Res

onan

ce u

nits

1800

1200

600

−600

0

0 50

g

f

e

c

ab

d

100 150

Time (s)

200

(b)

(a)

Figure 5.3 Validation of CaM as a target protein of HBC. (a) Surface plasmon resonanceanalysis of interaction between HBC and Ca2+/CaM. Purified Ca2+/CaM was immobilizedon a CM5 sensor chip and various concentrations of HBC were loaded into the sensorcell. Binding sensor grams were obtained from the BIAcore evaluation software. Kineticparameters of ka, kd ,KA, and KD are shown. (b) Docking model of HBC in a complexwith the C-terminal Ca2+/CaM domain. The docking mode of HBC (gray carbon) andW7 (orange carbon) obtained from FlexX. The Connolly molecular surface of the activesite is shown in purple with amino acid residues occupying the active site. Hydrogenatoms are not shown for clarity. The yellow dotted line indicates the hydrogen-bondinginteraction (d = 1.244 A). (From ref. 15.)

I: Competitive

(a) (b) (c)

−(KM)−1 (Vmax)−1

(v0)

−1

[S0]−1

I: UnCompetitive

Noncompetitive

Uninhibited

(v0)

−1

[S0]−1

I : KI = KI ’

I : KI > KI ’

I : KI < KI ’

(v0)

−1[S0]−1

Apparent KM Apparent Vmax Apparent KM Apparent Vmax

VmaxaKM Vmax /aKM

Apparent KM Apparent Vmax

Vmax /b

Vmax /b

KM /b

aKM /b

Vmax /baKM /bE E

S S

KI

I

I

I

S

SKI’

KI I KI’

E

−−

−

Figure 7.3 Inhibitor types, corresponding apparent kinetic constants, and reaction mech-anisms. Here α is defined as (1 + [I]/KI), while β is short for (1 + [I]/K ′

I). Note that therate equation for a given inhibitor type can be obtained simply by substituting the kineticconstants in equation (2) with the apparent constants.

Figure 7.6 Fragment-based design: When two optimized and independent ligands(square and triangle) are joined, the binding of the resulting compound to its target protein(red) is superior to that of separate fragments.

Figure 7.7 U Dock 1.1, in a search for an ideal ligand conformation. Note that this posewill probably be rejected since it clashes with the surface of the active site and protrudesinto the protein.

Sensitive or Synthetic-lethal

Resistant

Gene mutation array

Querycompound

Mut

ant1

Mut

ant2

Mut

ant3

Mut

ant4

Mut

ant5

Mut

ant6

Mut

ant7

Mut

ant8

Mut

ant9

Mut

ant1

0

Gen

e ar

ray

Gene A

Gene B

Gene C

Gene Z

Target proteinidentification

Mut

ant1

Mut

ant2

Mut

ant3

Mut

ant4

Mut

ant5

Mut

ant6

Mut

ant7

Mut

ant8

Mut

ant9

Mut

ant1

0

Gene mutation array

Com

poun

d ar

ray

Target pathwayidentification

Compound A

Compound B

Compound C

Compound Z

Mut

ant1

Mut

ant2

Mut

ant3

Mut

ant4

Mut

ant5

Mut

ant6

Mut

ant7

Mut

ant8

Mut

ant9

Mut

ant1

0

Gene mutation array

Compare(i) (ii)

Figure 11.2 From phenotype observation to target pathway and target protein identi-fication. Profiling of chemical genetic interactions is of help for identification of targetpathway and target protein. Once phenotypes of gene mutants for a query compound aredefined, modes of action of the compound can be expected from statistical analysis usingphenotype compendia or functional analysis using public databases. For example, themutants 6, 8, and 10 are sensitive (shown in red) to the query compound and the mutants3 and 7 are resistant (shown in green) to the query compound. (i) Comparison with thecompendia of chemical genetic interaction profiles reveals that the query compound has asimilar target pathway or modes of action with those of the compound A. (ii) Comparisonwith the synthetic lethal profiles shows that the gene A product is predicted to be a targetprotein of the query compound. This strategy is particularly useful for reverse genomicsapproaches using yeast.