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ANALYTICAL CURRENTS NMR-based screening on fast track Driven to search for new drugs, researchers have recently developed a slew of clever NMR methods for high-throughput screening of ligands that bind to important target biomolecules. Claudio Dalvit and colleagues at Pharmacia (Italy and the United States) describe a new NMR method that can handle a broad range of ligands, including the extremes of high-affinity ligands and molecules that bind covalently to receptors, which typically escape detection in methods that rely on an excess of ligand, and weakly soluble ligands, which are lost because of NMR’s poor sensitivity. Moreover, the new method estimates the ligand’s binding constant with a singlepoint measurement. The approach is based on competition binding experiments. Initially, a ref-
erence compound with the right NMR characteristics and a medium to low affinity for the target is chosen; the reference’s binding constant is determined by isothermal titration calorimetry. The authors then outline a series of steps and equations that lead to two types of graphs: the ratio of signal intensity for two of the reference ligand’s resonances versus the ratio of bound ligand to total ligand ([EL]/[LTOT]) and a plot that relates the NMR selective longitudinal relaxation rate (R1,s) to [EL]/[LTOT]. Experiments with R1,s for determining ligand binding are well known and require inversion of one of the reference’s resonances. Moreover, the R1,s approach for the ligand competition experiments can be done in an automated mode—a plus for high-throughput work.
Armed with their equations, data, and graphs, the researchers say that they can quickly screen compounds against targets ranging from proteins to DNA fragments to possibly plant extracts. They demonstrate the technique with tryptophan (Trp) as the reference and human serum albumin as the target. Screening against a chemical mixture, they found that diazepam effectively competes with Trp. The new estimate of diazepam’s binding constant is somewhat larger than the value in the literature, probably due to the presence of a second binding site on albumin for Trp. The limit to the method is that it only works with highto medium-affinity compounds that compete or have an allosteric effect on the reference ligand. (J. Am. Chem. Soc. 2002, 124, 7702–7709)
(a) 2.4 2.2
Nanotubes perform chiral separations
1/R1,s
2.0 1.8
To achieve chiral separations without
the antibody-modified nanotubes allow
1.6
chromatography, one option is selective
one enantiomer to pass to the other side.
1.4
transport through a membrane in which
The researchers tested the membranes
enantioselective molecules are embed-
using an antibody that preferentially binds
ded. Charles Martin and colleagues at the
the RS enantiomer of an inhibitor of the
University of Florida and VTT Biotechnolo-
enzyme aromatase. Membranes with a
gy (Finland) report the newest version of
35-nm pore diameter achieved an average
this approach: a synthetic bionanotube
transport selectivity coefficient (the ratio
membrane that incorporates enantiose-
of the RS flux to the SR flux) of ~2. For 20-
lective antibodies.
nm pores, the selectivity coefficient in-
1.2 1.0 0.00
0.02
0.04
0.06 0.08 [EL]/[LTOT]
0.10
0.12
IH4 /IH2
(b) 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.00
The membranes are based on alumina films laced with cylindrical pores. The re0.02
0.04
0.06 0.08 [EL]/[LTOT]
0.10
0.12
Screening for drug candidates. Top: Plot of 1/R1,s for a Trp resonance as a function of [EL]/ [LTOT]. Bottom: Ratio of signal intensities for two Trp C–H resonances from R1,s experiments as a function of the ratio [EL]/ [LTOT]. 454 A
creased to 4.5. The researchers addressed the prob-
searchers synthesize silica nanotubes
lem of the antibodies binding the target
within the pores and attach the antibodies
molecules irreversibly by adding the sol-
to the inner walls of the nanotubes. A
vent dimethyl sulfoxide to “tune” the anti-
racemic mixture of molecules on one side
body affinity. (Science, 2002, 296,
of the membrane is gradually purified as
2198–2200)
A N A LY T I C A L C H E M I S T R Y / S E P T E M B E R 1 , 2 0 0 2
