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Scheme 3.1 Isosteric competition diagram.

Slsurosporine K252a NGD-157 Trimethoprim

Fig. 3.10 Examples of isosteric binding competition. (A) ALIS-MS results for the titration of 5 ||M Zap-70 by staurosporine in the presence of a 5 m concentration of its structural congener K252a; and (B) titration of 5 | M DHFR with the known DHFR inhibitor trimethoprim in the presence of ligand NGD-157 at 5 |m concentration. Linear MS response ratios in these experiments are consistent with direct binding competition. (C) Compound structures.

Slsurosporine K252a NGD-157 Trimethoprim

Fig. 3.10 Examples of isosteric binding competition. (A) ALIS-MS results for the titration of 5 ||M Zap-70 by staurosporine in the presence of a 5 m concentration of its structural congener K252a; and (B) titration of 5 | M DHFR with the known DHFR inhibitor trimethoprim in the presence of ligand NGD-157 at 5 |m concentration. Linear MS response ratios in these experiments are consistent with direct binding competition. (C) Compound structures.

responses for two competing ligands will be linear as a function of increasing titrant.

As another example of direct binding competition, Fig. 3.10 shows competition profiles for the emerging immunosuppression target Zap-70 kinase [54] using staurosporine and its structural congener K252a, both well known ligands for active sites of nearly all protein kinases. As expected, these two structurally similar ligands yield a linear ratio of MS responses, consistent with direct binding competition. Though ATP has poor sensitivity in electrospray ionization MS, the Zap-70 example demonstrates that a known ATP-binding site inhibitor such as staurosporine can be used in ALIS as an ESI-MS-sensitive surrogate of ATP or other nucleotide ligand. As another example, and one that demonstrates the method for two compounds of very different structure, the DHFR ligand NGD-157 (whose Kd determination was shown previously in Fig. 3.8) is directly competitive with the known DHFR ligand trimethoprim [55] as shown by the linear response ratio plotted in Fig. 3.10B.

ALIS competition experiments can also demonstrate whether two ligands bind allosterically with respect to one another. Such allosteric binding can be positively cooperative, where binding by one ligand enhances binding by a second; or negatively cooperative, such that binding by the first diminishes binding by a second; or non-cooperative, so binding by one has no effect on the binding of another li-

138 | 3 ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization Scheme 3.2 Allosteric competition diagram.

gand. Scheme 3.2 shows the ternary complex model of allosteric binding [56]. In this model, ligands Si and S2 bind distinct sites on receptor E with dissociation constants Kd1 and Kd2, respectively. However, if both ligands bind simultaneously to the receptor, they may affect each other's binding constant by an amount described as the binding cooperativity factor, denoted here as a. For example, S1 binds to E with dissociation constant Kd1, but it also binds to the binary complex E S2 to form ternary complex E S1 S2 with dissociation constant a: Kd1. Where a > 1, allosteric interaction by one of the ligands increases the dissociation constant of the other, resulting in negative binding cooperativity. Where a < 1, positive cooperativity results, and if a = 1, binding by one ligand has no effect on the binding of the other [57].

ALIS cannot separate binary protein-ligand complexes from allosterically bound ternary complexes; all protein-ligand species co-elute from the SEC stage. The measured recovery of a particular ligand therefore represents the sum of the protein-ligand complexes containing that ligand. As a consequence, the ratio of the ALIS MS responses of a titrated competitor versus an allosteric ligand will not be a straight line, as was the case with direct competition; rather the ratio plot will be an asymptotically bound hyperbolic curve if the two ligands can form a ternary complex with the protein target.

The Akt-1 kinase ligand NGD-28835, discovered by ALIS screening of mass-encoded libraries against the basal form of its target [58], provides an example of allosteric binding interaction. As shown in the ALIS titration experiment for Akt-1 and NGD-28835 versus staurosporine in Fig. 3.11, the ALIS response for staur-osporine is diminished to a constant value while the titrant response plateaus as the receptor reaches saturation. This yields an asymptotically bound response ratio, indicating allosteric binding with respect to staurosporine and detection of a ternary complex of Akt-1, staurosporine, and NGD-28835 by ALIS. This result is consistent with NGD-28835 binding outside the ATP-binding pocket of Akt-1, and indicates negative binding cooperativity by NGD-28835.

Compounds that inhibit Akt-1 are of increasing interest as possible oncology therapeutics [59]. Akt-1 is a multi-domain protein that is known to be activated after binding of its pleckstrin homology (PH) domain to its endogenous target. A report from researchers at Merck indicates that their Akt-1 inhibitor does

Fig. 3.11 Examples of allosteric binding competition. Titration of 5 mM Akt-1 plus 5 mM staurosporine by: (A) NGD-28835 and (B) Merck-1 does not yield linear response ratios for the two competing ligands. Asymptotically bound response ratios indicate allosteric binding between these two ligands and staurosporine. (C) Titration of 5 mM Akt-1 plus 5 mM NGD-28835 by Merck-1 does yield a linear ratio of MS responses, indicating these two compounds bind the same site on Akt-1. (D) Compound structures.

Fig. 3.11 Examples of allosteric binding competition. Titration of 5 mM Akt-1 plus 5 mM staurosporine by: (A) NGD-28835 and (B) Merck-1 does not yield linear response ratios for the two competing ligands. Asymptotically bound response ratios indicate allosteric binding between these two ligands and staurosporine. (C) Titration of 5 mM Akt-1 plus 5 mM NGD-28835 by Merck-1 does yield a linear ratio of MS responses, indicating these two compounds bind the same site on Akt-1. (D) Compound structures.

not bind to the kinase domain; rather, it binds Akt-1 at its PH domain [60]. ALIS competition experiments between staurosporine and the Merck compound (Merck-1) indicate allosteric binding between these two ligands, as evidenced by the hyperbolic ratio plot of the ALIS responses in the titration experiment shown in Fig. 3.11B.

NGD-28835 and Merck-1 both bind allosterically with respect to staurosporine. To test whether NGD-28835 and Merck-1 bind the same site, ALIS competition experiments were conducted with these two compounds. Though they are structurally dissimilar, a linear ratio plot in Fig. 3.11C confirms isosteric binding for these two compounds, indicating that they both bind the PH domain and effect a biological response through this mechanism, rather than through traditional binding to the kinase active site. It is noteworthy that the ALIS competition method can discern the binding sites of ligands to the inactive form of a receptor (here, the basal form of a kinase), which is a challenging task using traditional biochemical assays.

140 | 3 ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization 3.5.2

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