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. 2022 Jul 29;8(30):eabo0696.
doi: 10.1126/sciadv.abo0696. Epub 2022 Jul 29.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy

Cristina Olivieri  1 , Geoffrey C Li  2 , Yingjie Wang  2   3 , Manu V S  1 , Caitlin Walker  1 , Jonggul Kim  2 , Carlo Camilloni  4 , Alfonso De Simone  5 , Michele Vendruscolo  6 , David A Bernlohr  1 , Susan S Taylor  7 , Gianluigi Veglia  1   2
Affiliations

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy

Cristina Olivieri et al. Sci Adv. .

Abstract

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

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Figures

Fig. 1.
Fig. 1.. Structure and conformational transitions of PKA-C upon binding ATP-competitive inhibitors.
(A) X-ray structure of PKA-C [Protein Data Bank (PDB): 1ATP]. The subdomains are colored using Hanks and Hunter convention (1). (B) C-spines, R-spines, and shell residues for the ATP-, balanol-, and H89-bound structures. (C) Chemical structures of ATP, balanol, and H89. The p-hydroxybenzamide group in balanol (or isoquinoline ring in H89) mimics the adenine moiety of ATP, the perhydroazepane ring in balanol (or sulfonamide in H89) occupies the ribose subsite, and the benzophenone group in balanol (or bromocinnamoyl group in H89) mimics the triphosphate of ATP. (D) Coordinate chemical shift behavior plots showing that the ATP-competitive inhibitors shift the distribution of the chemical shifts for the amides and methyl groups of PKA-C toward an intermediate state. PC, principal component. The amide and methyl chemical shift lists used for the analysis are based on Kim et al. (15).
Fig. 2.
Fig. 2.. Coordinated chemical shift changes of the kinase in response to ligand binding.
(A) CHESCA maps for the amides (top portion) and methyl groups (bottom portion) upon binding ATPγN, balanol, and H89. The chemical shift covariance was calculated using four states of PKA-C: apo, ADP bound, inhibitor bound, and inhibitor and PKI bound. Only correlation coefficients () > 0.98 are reported. Enlarged CHESCA maps are available in the Supplementary Materials. (B and C) Chemical shift correlation networks for (riJ) >0.98 mapped onto the PKA-C structure for amide and methyl groups, respectively. The color-coded spider plots indicate the extent of the correlations, with red being the highest and blue being the lowest. ATPγN shows the highest degree of chemical shift covariance between the two lobes of the enzyme. The number and extent of correlation become gradually lower going from balanol to H89. The amide and methyl chemical shift lists used for the analysis are derived from the spectra published by Kim et al. (15).
Fig. 3.
Fig. 3.. The density of the intramolecular allosteric network is linearly correlated with the degree of binding cooperativity.
The ζ score derived from the CHESCA analysis indicates the density of the chemical shift correlations as defined by Eq. (8), with normalization against ATPγN binding (ζ = 1.0). The cooperativity coefficient (σ) is derived from thermodynamics measurements and represents the ratios of the PKI-binding constants to PKA-C for in the absence and presence of nucleotides or ATP-competitive inhibitors. The σ value is from Kim et al. (15).
Fig. 4.
Fig. 4.. Dynamic response of the spine residues of PKA-C upon binding ATPγN and ATP-competitive inhibitors.
(A and B) Relaxation dispersion curves of the methyl side chain groups measured at 700 MHz (black) and 850 MHz (red). PKA-C shows substantial conformational exchange upon binding ATPγN, a reduced exchange with balanol, and almost no exchange with H89. Duplicate measurements were carried out at νCPMGvalues of 50, 500, and 1000 Hz. Errors were propagated and reported as error bars. (C) 13C CEST measured on a 900-MHz spectrometer using a B1 field of 15 Hz (red) and 30 Hz (blue). The methyl groups for L95 (R-spine), V104 (shell), and I150 (allosteric hotspot) show distinct conformationally excited states in the apo form. Binding of ATPγN, balanol, or H89 suppresses the conformationally excited states for these core residues. (D) Formation of new excited states for V80 and I85 (αB helix) for both ATPγN-bound and balanol-bound complexes and L173 (C-spine) for balanol-bound complex. The CEST profiles and the chemical shift difference for V80 and I85 for the ATPγN-bound and balanol-bound complexes are very similar. ppm, parts per million. (E to G) Mapping of the residues showing a change in the excited states (i.e., promotion, suppression, or broadening of the ground state) for PKA-C/ATPγN, PKA-C/balanol, and PKA-C/H89 complexes, respectively. CEST profiles were fit to a two-state exchange process (see Materials and Methods).
Fig. 5.
Fig. 5.. Relationship between conformational entropy and binding cooperativity.
(A) Mapping of the methyl group order parameters on the different structures of PKA-C bound to different ligands. For clarity, the ligands are omitted from the figure. (B) Linear relationship between the cooperativity coefficient (σ) and the conformational entropy (ΔSconf) of methyl side chains for the different ligated species of PKA-C. The ΔSconf values are calculated using Eq. 4 in Materials and Methods. The σ value is calculated from the isothermal titration calorimetry (ITC) data from Kim et al. (15).
Fig. 6.
Fig. 6.. Summary of the ATP-competitive ligands on correlated structural and dynamic changes.
ATPγN binding causes coordinated structural changes as reflected by the high value of σ, which is concomitant to a rigidification of the methyl group fast dynamics (ΔO2avg) throughout the entire enzyme, and an increase of methyl group slow dynamics (Rex). Balanol binding shows a decrease of σ value, an increase of ΔO2avg, and a decrease of Rex. Last, H89 shows the lowest σ value, the largest ΔO2avg, and almost no conformational exchange.
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