Distribution of a molecule within different tissues in the body is an important factor in the selection of drug candidates. Drug candidates which are highly bound to blood plasma proteins are unavailable to act on the target proteins, but are also protected from metabolism. This can either have a positive or negative effect, but either way, the degree to which a molecule is bound to serum proteins is increasingly recognized as an important decision-affecting metric even in the early stages of drug discovery.
Serum proteins are a diverse set of proteins. The most studied is human serum albumin (HSA), which binds many neutral and anionic molecules. Alpha(1)-acid glycoprotein (AGP) binds several cationic molecules, and lipoproteins bind mostly lipophilic molecules. Other proteins, such as globulins, rarely interact with molecules. The best way of measuring serum protein is by equilibrium dialysis with whole (100%) human sera. Diluted (50% or 10% serum) can be used, but results are less reliable.
Equilibrium dialysis involves a semi-permeable membrane which allows free small molecules, but not bound small molecules, to pass through. The chamber on one side includes human serum or human plasma, while the chamber on the other side includes only an isotonic buffer. The bound molecule is trapped in the protein chamber, while the free molecule equilibrates until the concentrations are equal on either side. The total amount of molecule on either side is then quantitated by LC-MS.
Figure 1: Measuring serum protein (orange, yellow circles) binding of small molecules (black) by equilibrium dialysis across a semi-permeable membrane (dashed).
We chose warfarin as a highly-bound control. First, we verified that the MS signal is linear with concentration across the concentration ranges. Next, the ratio of the MS signals in the buffer and protein chambers were separately quantitated, and the ratio used to calculate the percent bound. The literature values are 95-98%, and our results at 10 and 100 uM concentrations were in excellent agreement with that, but results at lower concentrations were increasingly variable.
|Warfarin concentration (uM)||Percent Bound (%)|
|100||98.0 ± 0.1|
|10||95 ± 2|
|1||71 ± 16|
Figure 2: Warfarin, a highly-bound control, is found at low concentrations in the buffer chamber (top), and at high concentrations in the serum chamber (bottom). The ratio of the areas is used to determine the percent binding.
Next, we chose captopril as a low-binding control. The literature values are 25-30%, and our results at 10 uM were in excellent agreement with that. In addition to a relative ratio, we also prepared several control samples of captopril to do an absolute quantitation. These data show that the sample is being fully recovered at the 10 uM level. Again, concentrations of 1 uM seem to produce highly variable results, both in the values for percent bound as well as recovery.
|Captopril concentration (uM)||Percent Bound (%)||Percent Recovery (%)|
|10||23 ± 6||96 ± 2|
|1||34 ± 20||73 ± 23|
Finally, we assayed several compounds with reported serum binding, all at the 10 uM level. Neomycin and oxacillin stock solutions were prepared 10 mM in water, while the others were prepared 10 mM in DMSO. This gives a final DMSO concentration of 0.1% for all the compounds except neomycin and oxacillin. Although the agreement between our method and literature values is not perfect, we can readily differentiate low-binding (neomycin, metronidazole, captopril) from moderately bound (dexamethasone, clarithromycin, oxacillin) and highly bound (warfarin, chlorpromazine, diclofenac) compounds.
|Compound (10 uM)||Percent Bound (%)||Literature Values (%)|
|dexamethasone||80 ± 4||70|
|clarithromycin||90 ± 5||70|
|oxacillin||89 ± 2||94.2 ± 2.1|
For each compound, only 50 uL of a 10 mM stock was required, which is amenable to most medicinal chemistry programs. If more precision is required and 100 mM stocks are available, higher concentrations can be used.
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