Reversible Covalent Inhibition of eEF‐2K by Carbonitriles

Devkota, Ashwini K.; Edupuganti, Ramakrishna; Cheng, Yan; Shi, Yue; Jose, Jiney; Wang, Qiantao; Kaoud, Tamer S.; Cho, Eun Jeong; Ren, Pengyu; Dalby, Kevin N.

doi:10.1002/cbic.201402321

Cited by 26 publications

(31 citation statements)

References 49 publications

(49 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…As mentioned previously, the CTD of eEF-2K closely associates with the KD and modulates its activity. A bioinformatics analysis 69 indicates that E717 coevolves with R200 on the KD, a residue predicted to be exposed in our previously reported homology model of the KD 29 . This could indicate that a possible E717-R200 salt-bridge provides a point of contact between the CTD and the KD, and that perhaps the E717A mutation alters the interface between the KD and CTD, thereby modulating kinase activity.…”

Section: Resultsmentioning

confidence: 93%

Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase

et al. 2016

Self Cite

View full text Add to dashboard Cite

Eukaryotic elongation factor 2 kinase (eEF-2K) phosphorylates its only known physiological substrate, elongation factor 2 (eEF-2), which reduces the affinity of eEF-2 for the ribosome and results in an overall reduction in protein translation rates. The C-terminal region of eEF-2K, that is predicted to contain several SEL-1 like helical repeats (SLRs), is required for the phosphorylation of eEF-2. Using solution NMR methodology, we have determined the structure of a 99-residue fragment from the extreme C-terminus of eEF-2K (eEF-2K627-725) that encompasses a region previously suggested to be essential for eEF-2 phosphorylation. eEF-2K627-725 contains four helices, of which the first (αI) is flexible, and does not pack stably against the ordered helical core formed by the last three helices (αII-αIV). The helical core shows significant structural similarity with members of the tetratricopeptide repeat (TPR) family that includes SLRs. The two penultimate helices, αII and αIII, comprise the TPR, and the final helix, αIV, appears to have a capping function. The eEF-2K627-725 structure illustrates that the C-terminal deletion that was shown to abolish eEF-2 phosphorylation does so by destabilizing αIV and therefore, the helical core. Indeed, mutation of two conserved C-terminal tyrosines (Y712A/Y713A) in eEF-2K previously shown to abolish eEF-2 phosphorylation, leads to the unfolding of eEF-2K627-725. Preliminary functional analyses indicate that neither a peptide encoding a region deemed crucial for eEF-2 binding, nor isolated eEF-2K627-725 inhibit eEF-2 phosphorylation by full-length eEF-2K. Taken together, our data suggest that the extreme C-terminal region of eEF-2K, in isolation, does not provide a primary docking site for eEF-2.

show abstract

Section: Resultsmentioning

confidence: 93%

Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…4D) and the behavior of similar inhibitors 49 for other proteins suggest that it may display slow reversibility. 49,56 This potential reversibility combined with the kinetic competition between crosslinking and polymerization might explain the unusual inhibitory properties of Remdesivir.…”

Section: Discussionmentioning

confidence: 99%

RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

Aranda

Orozco

2020

Preprint

View full text Add to dashboard Cite

We combine sequence analysis, molecular dynamics and hybrid quantum mechanics/molecular mechanics simulations to obtain the first description of the mechanism of reaction of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and of the inhibition of the enzyme by Remdesivir. Despite its evolutionary youth, the enzyme is highly optimized to have good fidelity in nucleotide incorporation and a good catalytic efficiency. Our simulations strongly suggest that Remdesivir triphosphate (the active form of drug) is incorporated into the nascent RNA replacing ATP, leading to a duplex RNA which is structurally very similar to an unmodified one. We did not detect any reason to explain the inhibitory activity of Remdesivir at the active site. Displacement of the nascent Remdesivir-containing RNA duplex along the exit channel of the enzyme can occur without evident steric clashes which would justify delayed inhibition. However, after the incorporation of three more nucleotides we found a hydrated Serine which is placed in a perfect arrangement to react through a Pinner's reaction with the nitrile group of Remdesivir. Kinetic barriers for crosslinking and polymerization are similar suggesting a competition between polymerization and inhibition. Analysis of SARS-CoV-2 mutational landscape and structural analysis of polymerases across different species support the proposed mechanism and suggest that virus has not explored yet resistance to Remdesivir inhibition.

show abstract

“…As no X-ray crystal structure for eEF2K is in the public domain, a homology model has been built in our group (Devkota et al, 2014 ) using the crystal structures of the alpha-kinase domain of myosin heavy chain kinase A (MHCKA, PDB ID: 3LKM) (Ye et al, 2010 ) and transient receptor potential (TRP) channels (ChaK) (PDB ID: 1IA9) (Yamaguchi et al, 2001 ). Based on this 3D model structure, compounds were docked into the ATP binding site of eEF2K using the ChemPLP (Korb et al, 2009 ) and Goldscore (Jones et al, 1995 , 1997 ) scoring functions in the GOLD5.1 software package.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we explored the possibility of using in silico approaches to design inhibitors for novel targets that do not have crystal structures. Based on a homology model of eEF2K that we built earlier (Devkota et al, 2014 ), three hypothetical binding poses of A-484954 were first generated from docking. The relative binding free energies of seven novel analogs of A-484954 were calculated for each hypothetical pose using alchemical free energy approach.…”

Section: Introductionmentioning

confidence: 99%

Using docking and alchemical free energy approach to determine the binding mechanism of eEF2K inhibitors and prioritizing the compound synthesis

Wang

Edupuganti

Tavares

et al. 2015

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

A-484954 is a known eEF2K inhibitor with submicromolar IC50 potency. However, the binding mechanism and the crystal structure of the kinase remains unknown. Here, we employ a homology eEF2K model, docking and alchemical free energy simulations to probe the binding mechanism of eEF2K, and in turn, guide the optimization of potential lead compounds. The inhibitor was docked into the ATP-binding site of a homology model first. Three different binding poses, hypothesis 1, 2, and 3, were obtained and subsequently applied to molecular dynamics (MD) based alchemical free energy simulations. The calculated relative binding free energy of the analogs of A-484954 using the binding pose of hypothesis 1 show a good correlation with the experimental IC50 values, yielding an r2 coefficient of 0.96 after removing an outlier (compound 5). Calculations using another two poses show little correlation with experimental data, (r2 of less than 0.5 with or without removing any outliers). Based on hypothesis 1, the calculated relative free energy suggests that bigger cyclic groups, at R1 e.g., cyclobutyl and cyclopentyl promote more favorable binding than smaller groups, such as cyclopropyl and hydrogen. Moreover, this study also demonstrates the ability of the alchemical free energy approach in combination with docking and homology modeling to prioritize compound synthesis. This can be an effective means of facilitating structure-based drug design when crystal structures are not available.

show abstract

Reversible Covalent Inhibition of eEF‐2K by Carbonitriles

Cited by 26 publications

References 49 publications

Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase

Structure of the C-Terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase

RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

Using docking and alchemical free energy approach to determine the binding mechanism of eEF2K inhibitors and prioritizing the compound synthesis

Contact Info

Product

Resources

About