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MCAT

Unit 3: Lesson 1

Chemical and physical sciences practice passage questions

Isothermal titration calorimetry in drug development

Problem

Isothermal Titration Calorimetry (ITC) is a procedure used in pharmaceutical research to identify lead compounds for therapeutic intervention and to optimize the affinity and selectivity of a drug candidate. Alternatively, ITC can be used to elucidate on enzyme kinetics or to confirm its mechanism of action. Unlike previous assays, ITC is more versatile and has applicability to a gamut of different ligands, i.e. proteins, lipids, DNA, ions, with no limit on molecular weight.
The major features of a typical isothermal titration calorimeter are the reference cell, sample cell, syringe, and adiabatic shield. Enclosed within the shield are the sample cell containing the protein target in buffer and the reference cell containing only the buffer. The syringe for adding titrant contains the compound or binding partner. During the experiment, constant power is supplied to the reference cell heater, while power is supplied to the sample cell feedback heater to bring the two samples back to the same temperature due to the difference induced by binding.
Figure 1. Schematic diagram of an isothermal titration calorimetry instrument

Attribution: Spinal83, CC-BY-SA 3.0.
At the end of the procedure, the following parameters can be attained: affinity constant (KD), enthalpy changes (ΔH), and binding stoichiometry (n). ∆S and ∆G are calculated via this relationship:
ΔG = ΔH - TΔS = RTlnKstart subscript, D, end subscript
where R is the gas constant and T is the absolute temperature. The binding energy can also be quantified by an enthalpic and entropic term. The enthalpic term (ΔH) is a measure of the changes in hydrogen and van der Waals bonding, and the entropic term (-TΔS), on the other hand, is an indication of changes in hydrophobic interaction and conformational changes.
A research group decides explore the role of two small molecule inhibitors, PES and VER-155008, both proposed to be Hsp70 inhibitors. The molecular chaperones of the Hsp70 family has been traditionally targeted for anti-cancer therapy. However, there are several paralogs of the Hsp70 proteins that require further investigation. Studies have shown that for significant reduction of cancer cell viability both knockdown of Hsp70 and Hsc70 are necessary. Below are the results from the experiment:
Figure 2. Isothermal titration calorimetry of the interaction of ADP, VER-155008, and PES to full-length human Hsp70. In the case of PES the titration was performed in the presence of ADP. Upper graph shows raw calorimetric data, and bottom graph showing integration of peaks with thermodynamic parameters indicated in center panel.

Attribution: Rainer Schlecht, CC-BY 4.0
In Figure 2, each dip represents an injection of the binding agent, and each subsequent injection produces a smaller energy change due to saturation of the macromolecule. After 45 minutes, with the continued injections in the ADP trial, what enthalpy is most likely observed?
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