Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium

Mizukoshi, Yumiko; Takeuchi, Koh; Tokunaga, Yuji; Matsuo, Hitomi; Imai, Masayuki; Fujisaki, Miwa; Kamoshida, Hajime; Takizawa, Takeshi; Hanzawa, Hiroyuki; Shimada, Ichio

doi:10.1126/sciadv.abd0480

Cited by 17 publications

(18 citation statements)

References 42 publications

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“…The conformational change in the protein required to enable access to did not occur at a sufficiently high rate for to outrank . NMR experiments have shown that the simple point mutation of F143W changes the redundancy of the P2 pocket to maintain the open state better 32 , which should lead to an increased association rate constant, k on , thus enabling the ligand to bind to Bcl-xL faster. Thus, taken together, such a mutation would most likely also decrease the population of configurations such as those found in 3INQ and .…”

Section: Discussionmentioning

confidence: 99%

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

Bekker

Fukuda

Higo

et al. 2021

Sci Rep

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We have performed multicanonical molecular dynamics (McMD) based dynamic docking simulations to study and compare the binding mechanism between two medium-sized inhibitors (ABT-737 and WEHI-539) that bind to the cryptic site of Bcl-xL, by exhaustively sampling the conformational and configurational space. Cryptic sites are binding pockets that are transiently formed in the apo state or are induced upon ligand binding. Bcl-xL, a pro-survival protein involved in cancer progression, is known to have a cryptic site, whereby the shape of the pocket depends on which ligand is bound to it. Starting from the apo-structure, we have performed two independent McMD-based dynamic docking simulations for each ligand, and were able to obtain near-native complex structures in both cases. In addition, we have also studied their interactions along their respective binding pathways by using path sampling simulations, which showed that the ligands form stable binding configurations via predominantly hydrophobic interactions. Although the protein started from the apo state, both ligands modulated the pocket in different ways, shifting the conformational preference of the sub-pockets of Bcl-xL. We demonstrate that McMD-based dynamic docking is a powerful tool that can be effectively used to study binding mechanisms involving a cryptic site, where ligand binding requires a large conformational change in the protein to occur.

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Section: Discussionmentioning

confidence: 99%

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

Bekker

Fukuda

Higo

et al. 2021

Sci Rep

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“…In other words, the protein surface can take on various structures according to its thermodynamic equilibrium under particular physiological conditions. In structural biology, the most abundant and stable population of a given protein structure is usually observed, while the rare and unstable structures are missed, although these can also be targeted by small molecules [ 70 , 71 , 72 ]. Because many proteins appear to lack druggable surface pockets, the concept of cryptic binding sites has received considerable attention, as this would expand the druggable proteome.…”

Section: Targeting Mysterious Pocketsmentioning

confidence: 99%

Druggable Transient Pockets in Protein Kinases

Umezawa

Kii

2021

Molecules

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Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

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“…To produce 15 N labeled hZα ADAR1 , BL21(DE3) bacteria were grown in M9 medium that contained 1 g/L 15 NH 4 Cl. The details of purification and expression of 15 N-labeled hZα ADAR1 proteins were described in a previous study [ 1 ]. The DNA and protein samples were dissolved in a 90% H 2 O/10% D 2 O NMR buffer containing 10 mM sodium phosphate (pH 6.0) with 100 mM NaCl salt.…”

Section: Methodsmentioning

confidence: 99%

“…Molecular motions play important roles in biological processes; however, their complexity makes the accurate identification of motions difficult. NMR spectroscopy is a powerful tool that had been used to characterize these motions according to the chemical exchanges and kinetic processes of molecules [ 1 , 2 , 3 ]. In particular, Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments can be used to provide information on protein folding and binding events, with protein motions reported in an ms-to-μs timescale [ 4 , 5 , 6 , 7 , 8 ].…”

Section: Introductionmentioning

confidence: 99%

“…The first step of CPMG analysis is the establishment of an appropriate model to determine the exchange process. The two-state exchange process is a simple kinetic model that has been widely used to understand molecular motions, such as enzyme catalysis, protein folding, molecular recognition, and allostery, using CPMG RD analysis [ 1 , 9 , 10 , 11 , 12 ]. Despite the fact that multi-state conformational exchanges including three-state transitions have also been studied using CPMG RD [ 13 , 14 , 15 , 16 , 17 ], elucidating the dynamics of more complicated motions of multi-state exchange processes with high accuracy remains challenging.…”

Section: Introductionmentioning

confidence: 99%

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Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

Lee

Choi

et al. 2021

IJMS

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Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

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Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium

Cited by 17 publications

References 42 publications

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

Cryptic-site binding mechanism of medium-sized Bcl-xL inhibiting compounds elucidated by McMD-based dynamic docking simulations

Druggable Transient Pockets in Protein Kinases

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

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