Understanding kinase‐inhibitor selectivity continues to be a major objective in kinase drug discovery. We probe the molecular basis of selectivity of an allosteric inhibitor (MSC1609119A‐1) of the insulin‐like growth factor‐I receptor kinase (IGF1RK), which has been shown to be ineffective for the homologous insulin receptor kinase (IRK). Specifically, we investigated the structural and energetic basis of the allosteric binding of this inhibitor to each kinase by combining molecular modeling, molecular dynamics (MD) simulations, and thermodynamic calculations. We predict the inhibitor conformation in the binding pocket of IRK and highlight that the charged residues in the histidine‐arginine‐aspartic acid (HRD) and aspartic acid‐phenylalanine‐glycine (DFG) motifs and the nonpolar residues in the binding pocket govern inhibitor interactions in the allosteric pocket of each kinase. We suggest that the conformational changes in the IGF1RK residues M1054 and M1079, movement of the ⍺C‐helix, and the conformational stabilization of the DFG motif favor the selectivity of the inhibitor toward IGF1RK. Our thermodynamic calculations reveal that the observed selectivity can be rationalized through differences observed in the electrostatic interaction energy of the inhibitor in each inhibitor/kinase complex and the hydrogen bonding interactions of the inhibitor with the residue V1063 in IGF1RK that are not attained with the corresponding residue V1060 in IRK. Overall, our study provides a rationale for the molecular basis of recognition of this allosteric inhibitor by IGF1RK and IRK, which is potentially useful in developing novel inhibitors with improved affinity and selectivity.
Amide−π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide−π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide−π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere−π interaction. We show that compared to the amide−π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea‐containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post‐translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.
more » « less- NSF-PAR ID:
- 10392272
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 1
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Molecular basis for differential recognition of an allosteric inhibitor by receptor tyrosine kinasesmore » « less
Abstract -
more » « less
The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named “Hv1 Inhibitor Flexibles” (HIFs), consist of two “prongs,” an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.
-
more » « less
Abstract The search for more effective and highly selective C–H bond oxidation of accessible hydrocarbons and biomolecules is a greatly attractive research mission. The elucidating of mechanism and controlling factors will, undoubtedly, help to broaden scope of these synthetic protocols, and enable discovery of more efficient, environmentally benign, and highly practical new C–H oxidation reactions. Here, we reveal the stepwise intramolecular SN2 nucleophilic substitution mechanism with the rate-limiting C–O bond formation step for the Pd(II)-catalyzed C(sp3)–H lactonization in aromatic 2,6-dimethylbenzoic acid. We show that for this reaction, the direct C–O reductive elimination from both Pd(II) and Pd(IV) (oxidized by O2oxidant) intermediates is unfavorable. Critical factors controlling the outcome of this reaction are the presence of the η3-(π-benzylic)–Pd and K+–O(carboxylic) interactions. The controlling factors of the benzylic vs ortho site-selectivity of this reaction are the: (a) difference in the strains of the generated lactone rings; (b) difference in the strengths of the η3-(π-benzylic)–Pd and η2-(π-phenyl)–Pd interactions, and (c) more pronounced electrostatic interaction between the nucleophilic oxygen and K+cation in the ortho-C–H activation transition state. The presented data indicate the utmost importance of base, substrate, and ligand in the selective C(sp3)–H bond lactonization in the presence of C(sp2)–H.
-
Abstract New drug production, from target identification to marketing approval, takes over 12 years and can cost around $2.6 billion. Furthermore, the COVID-19 pandemic has unveiled the urgent need for more powerful computational methods for drug discovery. Here, we review the computational approaches to predicting protein–ligand interactions in the context of drug discovery, focusing on methods using artificial intelligence (AI). We begin with a brief introduction to proteins (targets), ligands (e.g. drugs) and their interactions for nonexperts. Next, we review databases that are commonly used in the domain of protein–ligand interactions. Finally, we survey and analyze the machine learning (ML) approaches implemented to predict protein–ligand binding sites, ligand-binding affinity and binding pose (conformation) including both classical ML algorithms and recent deep learning methods. After exploring the correlation between these three aspects of protein–ligand interaction, it has been proposed that they should be studied in unison. We anticipate that our review will aid exploration and development of more accurate ML-based prediction strategies for studying protein–ligand interactions.more » « less
-
more » « less
Abstract Intramembrane-cleaving proteases (I-CLiPs) play crucial roles in physiological and pathological processes, such as Alzheimer’s disease and cancer. However, the mechanisms of substrate recognition by I-CLiPs remain poorly understood. The aspartic I-CLiP presenilin is the catalytic subunit of the γ-secretase complex, which releases the amyloid-β peptides (Aβs) through intramembrane proteolysis of the transmembrane domain of the amyloid precursor protein (APPTM). Here we used solution NMR to probe substrate docking of APPTM to the presenilin homologs (PSHs) MCMJR1 and MAMRE50, which cleaved APPTM in the NMR tube. Chemical shift perturbation (CSP) showed juxtamembrane regions of APPTM mediate its docking to MCMJR1. Binding of the substrate to I-CLiP decreased the magnitude of amide proton chemical shifts δHat the C-terminal half of the substrate APPTM, indicating that the docking to the enzyme weakens helical hydrogen bonds and unwinds the substrate transmembrane helix around the initial ε-cleavage site. The APPTM V44M substitution linked to familial AD caused more CSP and helical unwinding around the ε-cleavage site. MAMRE50, which cleaved APPTM at a higher rate, also caused more CSP and helical unwinding in APPTM than MCMJR1. Our data suggest that docking of the substrate transmembrane helix and helical unwinding is coupled in intramembrane proteolysis and FAD mutation modifies enzyme/substrate interaction, providing novel insights into the mechanisms of I-CLiPs and AD drug discovery.
