Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy

Eukaryotic elongation factor 2 kinase (eEF2K or Ca²⁺/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure–activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.     

Interaction of the σ2 Receptor Ligand PB28 with the Human Nucleosome: Computational and Experimental Probes of Interaction with the H2A/H2B Dimer

Sigma‐2 (σ₂) binding sites are an emerging target for anti‐neoplastic agents due to the strong apoptotic effect exhibited by σ₂ agonists in vitro and the overexpression of these sites in tumor cells. Nonetheless, no σ₂ receptor protein has been identified. Affinity chromatography using the high‐affinity σ₂ ligand PB28 and human SK‐N‐SH neuroblastoma cells was previously utilized to identify σ₂ ligand binding proteins, specifically histones H1, H2A, H2B, and H3.3a. To rationalize this finding, homology modeling and automated docking studies were employed to probe intermolecular interactions between PB28 and human nucleosomal proteins. These studies predicted interaction of PB28 with the H2A/H2B dimer at a series of sites previously found to be implicated in chromatin compaction and nucleosomal assembly. To experimentally verify this prediction, a competitive binding assay was performed on the reconstituted H2A/H2B dimer using [³H]PB28 as radioligand, and an IC₅₀ value of 0.50 nM was determined for PB28 binding. In addition, [³H]PB28 was found to accumulate with up to a fivefold excess in nuclear fractions over cytosolic fractions of SK‐N‐SH and MCF7 cells, indicating that PB28 is capable of entering the nucleus to interact with histone proteins. In conjunction with computational results, these data suggest that PB28 may exert its cytotoxic effect through direct interaction with nuclear material.     

Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium‐Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment

Dysregulation of Ca²⁺‐binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca²⁺‐bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca²⁺ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift‐based secondary‐structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow‐timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca²⁺‐coordinating EF‐hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca²⁺ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine‐tuning of its protein–protein interactions inside the cell during Ca²⁺ signaling.     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.     

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser⁷⁸⁵ and Ser⁷⁸⁷, that were similar in location and context to two amino acids of the Arabidopsis Na⁺/H⁺ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.     

Altered ionization of the B13 Glu in insulin B9 and B10 mutants: a computational analysis

An experimentally determined pK(a) change of +2.50 units has been reported for the B13 Glu residue in a dimeric B9 Ser --> Asp insulin mutant relative to the native dimer. Poisson-Boltzmann electrostatics-based pK(a) calculations were performed to probe the effect of the B9 Ser --> Asp and B10 His --> Asp mutations on aggregation and the ionization behaviour of the B13 carboxylate. The method produced shifts of +2.64 and +2.45 units for the pK(a) shift of the two B13 residues in the B9 mutant dimer relative to the wild-type dimer, which is in good agreement with the experimental value (<6% error). The calculations also suggest that the reason neither mutant insulin can aggregate into hexamers is the resultant crowding of negatively charged groups in the central solvent channel on hexamer formation. In the wild-type insulin, binding of zinc ions by B10 His overcomes this problem, whereas in the B10 mutant this possibility is ruled out by the absence of the zinc binding site. A series of mutations are predicted to stabilize the medically relevant, monomeric form of insulin.     

Protein adsorption in two-dimensional electrochromatography packed with superporous and microporous cellulose beads

Anion-exchange superporous cellulose (DEAE-SC) and microporous cellulose (DEAE-MC) adsorbents were packed in an electrochromatographic column, and the effect of external electric field (eEF) on the dynamic adsorption was investigated. The column was designed to provide longitudinal, transverse or 2-dimensional (2D) eEF. It was found that the electro-kinetic effect caused by the introduction of an electric field played an important role in the dynamic adsorption of bovine serum albumin to the adsorbents. The dynamic binding capacity (DBC) in the presence of 2D eEF was higher than in the presence of a one-dimensional eEF. The effect of flow velocity on the DBC of the two adsorbents was also demonstrated. It was found that the effect of electric field on the DEAE-MC column was more remarkable than that on the DEAE-SC column at the same flow rate, whereas the DEAE-SC column showed higher DBC and adsorption efficiency (AE) than the DEAE-MC column. With increasing flow rate, the DEAE-SC column could still offer high DBC and AE in the presence of the 2D eEF. For example, a DBC of 21.4 mg/mL and an AE of 57.7% were obtained even at a flow rate as high as 900 cm/h. The results indicate that the 2D electrochromatography packed with the superporous cellulose adsorbent is promising for high-speed protein chromatography.     

Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain

Ricin A-chain (RTA) is an N-glycosidase which removes a specificadenine residue from the large rRNA of eukaryotic ribosomes.As a consequence, the ribosome is inactivated and protein synthesisis inhibited leading to cell death. This report describes theeffects on enzyme activity of specific mutations of the conservedactive site Glu177. The activity of mutant proteins was initiallyscreened using an in vitro translation system. It was foundthat mutagenesis of Glu177 to Lys led to an apparent total inactivationof the enzyme, Glu177 to Ala had a small effect on activity,whereas the conservative Glu177 to Asp mutation had a significanteffect. The properties of Glu177 to Asp were investigated moreclosely. Mutant protein was purified from an Escherichia coliexpression system and kinetic analysis of the depurination activityassessed using salt-washed yeast ribosomes. It was shown thatthe K, of the mutant protein was unchanged when compared todata of wild type RTA; however, the k_cat was significantly decreased(49-fold compared to wild type RTA). This suggests that Glu177plays a predominant role in the rate-limiting step of the enzymaticmechanism and not in substrate binding. These data are discussedin relation to other reports of ricin Glu177 substitutions.     

Chaperone Activity of Bicyclic Nojirimycin Analogues for Gaucher Mutations in Comparison with N‐(n‐nonyl)Deoxynojirimycin

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp² iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp² iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.     

Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three‐dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c‐type cytochrome‐based chimeric proteins have been constructed and the domains swapped. According to X‐ray crystallographic analysis, the two chimeric proteins formed a domain‐swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H₂O coordinated hemes was formed. Binding of an oxygen molecule to the His/H₂O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe?O₂ stretching band was observed at 580 cm^?1 for the reduced/oxygenated heterodimer (at 554 cm^?1 under an ¹⁸O₂ atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.     

The HP1a disordered C terminus and chromo shadow domain cooperate to select target peptide partners

Drosophila melanogaster heterochromatin protein 1a (HP1a) is essential for compacted heterochromatin structure and the associated gene silencing. Its chromo shadow domain (CSD) is well known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a non-histone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres, and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization, and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics, and dimerization constant. We found that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive the homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a.     

Small-Molecule Activators of Glucose-6-phosphate Dehydrogenase (G6PD) Bridging the Dimer Interface

We recently identified AG1, a small-molecule activator that functions by promoting oligomerization of glucose-6-phosphate dehydrogenase (G6PD) to the catalytically competent forms. Biochemical experiments indicate that the activation of G6PD by the original hit molecule (AG1) is noncovalent and that one C₂-symmetric region of the G6PD homodimer is important for ligand function. Consequently, the disulfide in AG1 is not required for activation of G6PD, and a number of analogues were prepared without this reactive moiety. Our study supports a mechanism of action whereby AG1 bridges the dimer interface at the structural nicotinamide adenine dinucleotide phosphate (NADP⁺) binding sites of two interacting G6PD monomers. Small molecules that promote G6PD oligomerization have the potential to provide a first-in-class treatment for G6PD deficiency. This general strategy could be applied to other enzyme deficiencies in which control of oligomerization can enhance enzymatic activity and/or stability.     

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

Inhibition of Leishmania infantum Trypanothione Reductase by Azole‐Based Compounds: a Comparative Analysis with Its Physiological Substrate by X‐ray Crystallography

Herein we report a study aimed at discovering a new class of compounds that are able to inhibit Leishmania donovani cell growth. Evaluation of an in‐house library of compounds in a whole‐cell screening assay highlighted 4‐((1‐(4‐ethylphenyl)‐2‐methyl‐5‐(4‐(methylthio)phenyl)‐1H‐pyrrol‐3‐yl)methyl)thiomorpholine (compound 1 ) as the most active. Enzymatic assays on Leishmania infantum trypanothione reductase (LiTR, belonging to the Leishmania donovani complex) shed light on both the interaction with, and the nature of inhibition by, compound 1 . A molecular modeling approach based on docking studies and on the estimation of the binding free energy aided our rationalization of the biological data. Moreover, X‐ray crystal structure determination of LiTR in complex with compound 1 confirmed all our results: compound 1 binds to the T(SH)₂ binding site, lined by hydrophobic residues such as Trp21 and Met113, as well as residues Glu18 and Tyr110. Analysis of the structure of LiTR in complex with trypanothione shows that Glu18 and Tyr110 are also involved in substrate binding, according to a competitive inhibition mechanism.     

Mutational analysis of the N-capping box of the {alpha}-helix of chymotrypsin inhibitor 2

The N-terminus of the helix of the chymotrypsin inhibitor 2from barley (CI2) has an N-capping box (Ser at the first positionin the helix and Glu at position 4) as well as a frequentlyfound Glu at position 3. The energetic importance of this motifhas been studied by determining the free energy of unfoldingof the wild-type and protein mutants derived from those residuesusing guanidinium chloride-induced denaturation and differentialscanning microcalorimetry. Mutating N-cap residue Ser31 to eitherAla or Gly destabilizes CI2 by 0.8-1 kcal mol^–1. Truncationof the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabilizesthe protein by 1.5–2 kcal mol^–1. The N-capping boxis an important motif in stabilizing proteins and delineatingthe beginning of -helices in the pathway of protein folding.     

Chemical Synthesis of A Pore‐Forming Antimicrobial Protein,Caenopore‐5, by Using Native Chemical Ligation at a Glu‐Cys Site

The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore‐5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore‐forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid‐phase peptide synthesis and native chemical ligation. No γ‐linked by‐product was observed despite the use of a C‐terminal Glu‐thioester. The folding of the synthetic protein was confirmed by ¹H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore‐5. The permeabilisation activities of the protein and of shortened analogues were evaluated.     

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans

Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.