Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

The intrinsically disordered N-terminus of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is a critical ß-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the ß-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.

Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors.

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 µg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

Discovery of holoenzyme-disrupting chemicals as substrate-selective CK2 inhibitors.

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2ß subunits is essential for substrate selectivity. The CK2α/CK2ß interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2ß interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

Modification and Biological Evaluation of a Series of 1,5-Diaryl-1,2,4-triazole Compounds as Novel Agents against Pancreatic Cancer Metastasis through Targeting Myoferlin.

Pancreatic cancer is one of the most common cancers with an extremely low survival rate. Metastasis, as one of the key reasons of cancer-related death, is found in more than 50% pancreatic cancer patients at diagnosis. Novel therapeutic targets and drugs blocking cancer metastasis are urgently needed. Herein, we report a series of 1,5-diaryl-1,2,4-triazole derivatives as potent antimetastatic agents. Lead compound 6y displayed effective antimetastatic activities in pancreatic cancer in vitro and in vivo. Concomitant studies indicated that 6y probably binds with myoferlin (MYOF), a novel potential antitumor metastasis target, which regulates vesicle trafficking and metastasis-related proteins. Subsequent biophysical and biochemical methods verified that 6y bound to MYOF. Mechanism studies revealed that 6y inhibited pancreatic cancer metastasis through reversing the epithelial mesenchymal transition, inhibiting the secretions of matrix metalloproteinase and blocking the receptor tyrosine kinases. Our findings suggest that targeting MYOF with 6y may be a promising therapeutic strategy to prevent pancreatic cancer metastasis.

2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 1. Identification of an Allosteric Binding Site.

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach.

We used a combined approach based on fragment-based drug design (FBDD) and in silico methods to design potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), which has been recognized as an important therapeutic target in hematological cancers. Two subgroups of small compounds (including adenine and biaryl moieties) were identified as cN-II binders and a fragment growing strategy guided by molecular docking was considered. Five compounds induced a strong inhibition of the 5'-nucleotidase activity in vitro, and the most potent ones were characterized as noncompetitive inhibitors. Biological evaluation in cancer cell lines showed synergic effect with selected anticancer drugs. Structural studies using X-ray crystallography lead to the identification of new binding sites for two derivatives and of a new crystal form showing important domain swapping. Altogether, the strategy developed herein allowed identifying new original noncompetitive inhibitors against cN-II that act in a synergistic manner with well-known antitumoral agents.

A combination of spin diffusion methods for the determination of protein-ligand complex structural ensembles.

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.

Ligand specificity in fragment-based drug design.

Fragment-based drug design consists of identifying low-molecular weight compounds that weakly bind to a target macromolecule and will then be modified or linked to yield potent inhibitors. The specificity of these low-complexity and low-affinity molecules has rarely been discussed in the literature. To address this question, NMR spectroscopy was used to investigate the interactions of 150 fragments with five proteins: three proteins from the Bcl-2 family (Bcl-x(L), Bcl-w, and Mcl-1), human peroxiredoxin 5, for which very few ligands have been reported, and human serum albumin, which is known to bind a large number of ligands. Our results show that the fragments are rather versatile binders and able to identify binding hot spots in very different targets. Despite the different hit rates observed related to the druggability of the proteins, two scaffolds appear as preferred binders for all proteins. Low specificity was observed between homologous proteins or unrelated poorly druggable proteins, while higher specificity could be achieved with highly druggable targets.

Discovery of fragment molecules that bind the human peroxiredoxin 5 active site.

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.

NMR screening applied to the fragment-based generation of inhibitors of creatine kinase exploiting a new interaction proximate to the ATP binding site.

Using an in-house fragment NMR library, we identified a set of ligands that bind rabbit muscular creatine kinase, an enzyme involved in key ATP-dependent processes. The ligands docked to the crystal structures of creatine kinase indicated that a phenylfuroic acid could enter into a pocket adjacent to the nucleotide binding site. This fragment served as an anchor to develop in silico a series of potential inhibitors which could partly access the nucleotide binding site. The short synthesis of only four derivatives provided entirely novel hit compounds that reversibly inhibit creatine kinase at micromolar concentrations with a mixed ATP-competitive/noncompetitive mechanism in agreement with the structural model of the inhibited enzyme. These initial biologically active compounds are novel and modular and exploit a new interaction proximate to the ATP binding site.

Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer.

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.

Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR.

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.