Implementation of a photoaffinity pulldown probe

In our studies of DQ inhibitors, we observed inhibited cell proliferation, inhibited autophagy, and blocked mTOR signaling in DQ661 treated melanoma cells. The DQ661- P, 149, treated cells were assayed for autophagy inhibition and mTOR signaling.

Treatment of melanoma cells with DQ661-P 149 caused the accumulation of LC3B,

which indicated that autophagy was inhibited. Western blotting for 4E-BP1 and S6 demonstrated that phosphorylation was decreased upon treatment of A375P melanoma cells with 149, indicating that mTOR activity was suppressed. Therefore, we reasoned

that 149 would be effective in competitive PAL experiments.

Figure 3.14: Western blot assaying autophagy inhibition (LC3B) and mTOR signaling (p4E-BP1 and pS6) in DQ661-P 149 treated cells. Decreased p4E-BP1 and pS6 indicates inhibition of mTOR signaling.

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Doses for the pulldown assay were determined with maximum target capture in mind, while minimizing background non-specific binding. In a standard model for protein-substrate binding, the affinity of an enzyme for a substrate or inhibitor can be quantified in terms of the dissociation constant (Kd) of the ligand, defined by the

concentration of free enzyme and free ligand divided the concentration of enzyme-ligand complex. Utilizing this metric, a smaller numerical value indicates a tighter binding interaction. The amount of inhibitor present at concentrations below the Kd is rapidly complexed with enzyme. Enzymes quickly reach a point of saturation where no further ligand is bound after the dissociation constant is reached (Figure 3.15). Inhibitor added beyond the enzyme saturation point will have a greater contribution to unwanted

background signal than increased target binding. We hypothesized that for a phenotype to be induced, significant quantities of the target enzyme must be bound. Therefore, a dose of 200 nM was selected, because at this dose LC3B is observed to accumulate, indicating autophagy inhibition. From this result we hypothesized that a significant quantity of the putative target must be bound to cause the autophagy inhibition phenotype. Our proposed proteomic analysis was used only for protein identification purposes, and therefore we valued background signal reduction greater than increased target binding.

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The experimental controls are critical to the successful design of a pulldown experiment. At its core, the goal of a photoaffinity pulldown is to make an unbiased crosslink between the photoreactive functional group and the nearest protein to the probe. In all cases, addition of the probe functionality to parent warhead inherently will add significant non-specific binding, because desthiobiotin will be bound by native biotin binding proteins and benzophenone will make non-specific hydrophobic interactions with proteins. Further, it is expected that all small molecule therapeutics have non-specific protein binding due to the drug partitioning between the aqueous solvent and the more hydrophobic macromolecules of the cell.

The experimental setup becomes a balancing act of generating and isolating as many crosslinked potential targets as possible, while discerning which of those crosslinks are specific and which are non-specific. We utilized three controls for every probe tested.

Figure 3.15: A representation of a dissociation constant with an indication of enzyme saturation, where additional ligand would bind background non-specific binding rather than the target protein.

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This first control was a negative control in which cells were treated with the photoaffinity probe but were not exposed to UV irradiation. Proteins identified should not be covalent adducts and were regarded as nonspecific binding. In the next experiment, the cells were treated with the photoaffinity probe and irradiated with UV light. The benzophenone and the affinity handle will generate false targets which bind the photoaffinity probe but would not have significant affinity for the parent inhibitor. In order to separate these false positive results from the true potential targets, a competition control was utilized. We hypothesized that the mechanistic target of dimeric lysosomal inhibitors will bind equally or more potently to the parent inhibitor than the photoaffinity probe. This hypothesis was based on the observation that neither benzophenone nor biotin/desthiobiotin have been reported as inhibitors of autophagy. We hypothesized that the administration of a competition dose of tenfold greater magnitude than the photoaffinity probe would displace the photoaffinity probe from the putative protein target. This decrease in signal for proteins would indicate that their crosslinking was conferred by a specific binding affinity between the inhibitor warhead and the protein. The crosslinking of proteins which non-specifically bind the benzophenone or affinity handle should not be significantly altered by the competition dose.

Once cells were either irradiated or not irradiated, they were immediately harvested and lysed. The cell lysate was frozen at -80 °C in the presence of broad spectrum protease inhibitors until affinity purification. To facilitate affinity

chromatography, a streptavidin protein conjugated to polymer support was required. Neutravidin (Thermo Scientific), a bioengineered homolog of avidin and streptavidin,

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was chosen for our experiment. Neutravidin binds biotin as tightly as both avidin and streptavidin, but the surface of the protein is mutated to maximally reduce non-specific protein binding to the resin. The protein is conjugated to agarose as a solid support. The neutravidin resin was equilibrated with cell lysis buffer containing protease inhibitors, and was mixed with cell lysate for a period of 12-16 hours at 4 °C. The resin was then separated via centrifugation and washed with a series of NP-40-containing buffers to remove cell debris and non-specifically bound proteins. The washes were analyzed via SDS-PAGE to determine whether further washing was required.

The use of desthiobiotin as an affinity handle facilitated a non-denaturing elution step, yielding a cleaner elution from the resin. Desthiobiotin 131 (Figure 3.5) is an analog

of biotin 130 (Figure 3.5), where the sulfur atom is removed from the

tetrahydrothiophene ring, leaving only the cyclic urea of the fused ring system. Multiple orthogonal elution methods based on chemical linkers which are labile to bioorthogonal chemistry have been invented.108 _{We elected to utilize desthiobiotin because of synthetic} ease and the predefined protocols for elution from avidin resins.

Desthiobiotin binds avidin/streptavidin with a Kd of ~10-13 versus the Kd of biotin~10-15. The difference of two order of magnitude allows for elution of desthiobiotinylated molecules via incubation with a biotin-containing buffer. A 15- minute incubation of the solid supported neutravidin resin in a 4 mM biotin buffer produced proteomic profiles which were analyzed by trypsin digest LC-MS/MS to identify the proteins which were pulled down.

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