Vagelis Rinotas

A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF

Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumor necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis (ARO), whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach using N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we generated a novel mouse model of ARO caused by a new loss-of-function allele of Rankl with a glycine-to-arginine mutation at codon 278 (G278R) at the extracellular inner hydrophobic F β-strand of RANKL. Mutant mice develop severe osteopetrosis similar to Rankl-deficient mice, whereas exogenous administration of recombinant RANKL restores osteoclast formation in vivo. We show that RANKL(G278R) monomers fail to assemble into homotrimers, are unable to bind and activate the RANK receptor and interact with wild-type RANKL exerting a dominant-negative effect on its trimerization and function in vitro. Since G278 is highly conserved within the TNF superfamily, we identified that a similar substitution in TNF, G122R, also abrogated trimerization, binding to TNF receptor and consequently impaired TNF biological activity. Notably, SPD304, a potent small-molecule inhibitor of TNF trimerization that interacts with G122, also inhibited RANKL activity, suggesting analogous inhibitory mechanisms. Our results provide a new disease model for ARO and identify a functional amino acid in the TNF-like core domain essential for trimer formation both in RANKL and in TNF that could be considered a novel potential target for inhibiting their biological activities.

Novel Genetic Models of Osteoporosis by Overexpression of Human RANKL in Transgenic Mice

Receptor activator of NF‐κB ligand (RANKL) plays a key role in osteoclast‐induced bone resorption across a range of degenerative bone diseases, and its specific inhibition has been recently approved as a treatment for women with postmenopausal osteoporosis at high or increased risk of fracture in the United States and globally. In the present study, we generated transgenic mice (TghuRANKL) carrying the human RANKL (huRANKL) genomic region and achieved a physiologically relevant pattern of RANKL overexpression in order to establish novel genetic models for assessing skeletal and extraskeletal pathologies associated with excessive RANKL and for testing clinical therapeutic candidates that inhibit human RANKL. TghuRANKL mice of both sexes developed early‐onset bone loss, and the levels of huRANKL expression were correlated with bone resorption and disease severity. Low copy Tg5516 mice expressing huRANKL at low levels displayed a mild osteoporotic phenotype as shown by trabecular bone loss and reduced biomechanical properties. Notably, overexpression of huRANKL, in the medium copy Tg5519 line, resulted in severe early‐onset osteoporosis characterized by lack of trabecular bone, destruction of the growth plate, increased osteoclastogenesis, bone marrow adiposity, increased bone remodeling, and severe cortical bone porosity accompanied by decreased bone strength. An even more severe skeletal phenotype developed in the high copy Tg5520 founder with extensive soft tissue calcification. Model validation was further established by evidence that denosumab, an antibody that inhibits human but not murine RANKL, fully corrected the hyper‐resorptive and osteoporotic phenotypes of Tg5519 mice. Furthermore, overexpression of huRANKL rescued osteopetrotic phenotypes of RANKL‐defective mice. These novel huRANKL transgenic models of osteoporosis represent an important advance for understanding the pathogenesis and treatment of high‐turnover bone diseases and other disease states caused by excessive RANKL.

A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization, and Lymphoid Abnormalities in Mice

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-α/SPD304 Study

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a K d of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins.

Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)

We present an in silico drug discovery pipeline developed and applied for the identification and virtual screening of small-molecule Protein-Protein Interaction (PPI) compounds that act as dual inhibitors of TNF and RANKL through the trimerization interface. The cheminformatics part of the pipeline was developed by combining structure–based with ligand–based modeling using the largest available set of known TNF inhibitors in the literature (2481 small molecules). To facilitate virtual screening, the consensus predictive model was made freely available at: http://enalos.insilicotox.com/TNFPubChem/. We thus generated a priority list of nine small molecules as candidates for direct TNF function inhibition. In vitro evaluation of these compounds led to the selection of two small molecules that act as potent direct inhibitors of TNF function, with IC50 values comparable to those of a previously-described direct inhibitor (SPD304), but with significantly reduced toxicity. These molecules were also identified as RANKL inhibitors and validated in vitro with respect to this second functionality. Direct binding of the two compounds was confirmed both for TNF and RANKL, as well as their ability to inhibit the biologically-active trimer forms. Molecular dynamics calculations were also carried out for the two small molecules in each protein to offer additional insight into the interactions that govern TNF and RANKL complex formation. To our knowledge, these compounds, namely T8 and T23, constitute the second and third published examples of dual small-molecule direct function inhibitors of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.

A statistical approach for optimization of RANKL overexpression in Escherichia coli: Purification and characterization of the protein

Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3mM, a post-induction temperature of 25°C and a post-induction time of 6.5h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1 ± 0.5 μM.

Designer Xanthone: An Inhibitor Scaffold for MDR-Involved Human Glutathione Transferase Isoenzyme A1-1

Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC50 values (7: 1.59 ± 0.25 µM, 8: 5.30 ± 0.30 µM, and 17: 8.56 ± 0.14 µM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 µM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC50 = 10.54 ± 2.41 µM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC50 = 151.3 ± 16.3 µM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs.

Current Status and Future Prospects of Small–molecule Protein–protein Interaction (PPI) Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)

The overexpression of Tumor Necrosis Factor (TNF) is directly related to the development of several autoimmune diseases, such as rheumatoid and psoriatic arthritis, inflammatory bowel disease, Crohns disease, refractory asthma, and multiple sclerosis. Receptor Activator of Nuclear Factor Kappa- B Ligand (RANKL) belongs to the TNF family and is the primary mediator of osteoclast-induced bone resorption through interaction with its receptor RANK. The function of RANKL is physiologically inhibited by the action of osteoprotegerin (OPG), which is a decoy receptor that binds to RANKL and prevents the process of osteoclastogenesis. Malfunction among RANK/RANKL/OPG can also result in bone loss diseases, including postmenopausal osteoporosis, rheumatoid arthritis, bone metastasis and multiple myeloma. To disrupt the unwanted functions of TNF and RANKL, current attempts focus on blocking TNF and RANKL binding to their receptors. In this review, we present the research efforts toward the development of low-molecular-weight pharmaceuticals that directly block the detrimental actions of TNF and RANKL.

Transgenic Mice Carrying GLUD2 as a Tool for Studying the Expressional and the Functional Adaptation of this Positive Selected Gene in Human Brain Evolution

Human evolution is characterized by brain expansion and up-regulation of genes involved in energy metabolism and synaptic transmission, including the glutamate signaling pathway. Glutamate is the excitatory transmitter of neural circuits sub-serving cognitive functions, with glutamate-modulation of synaptic plasticity being central to learning and memory. GLUD2 is a novel positively-selected human gene involved in glutamatergic transmission and energy metabolism that underwent rapid evolutionary adaptation concomitantly with prefrontal cortex enlargement. Two evolutionary replacements (Gly456Ala and Arg443Ser) made hGDH2 resistant to GTP inhibition and allowed distinct regulation, enabling enhanced enzyme function under high glutamatergic system demands. GLUD2 adaptation may have contributed to unique human traits, but evidence for this is lacking. GLUD2 arose through retro-positioning of a processed GLUD1 mRNA to the X chromosome, a DNA replication mechanism that typically generates pseudogenes. However, by finding a suitable promoter, GLUD2 is thought to have gained expression in nerve and other tissues, where it adapted to their particular needs. Here we generated GLUD2 transgenic (Tg) mice by inserting in their genome a segment of the human X chromosome, containing the GLUD2 gene and its putative promoter. Double IF studies of Tg mouse brain revealed that the human gene is expressed in the host mouse brain in a pattern similar to that observed in human brain, thus providing credence to the above hypothesis. This expressional adaptation may have conferred novel role(s) on GLUD2 in human brain. Previous observations, also in GLUD2 Tg mice, generated and studied independently, showed that the non-redundant function of hGDH2 is markedly activated during early post-natal brain development, contributing to developmental changes in prefrontal cortex similar to those attributed to human divergence. Hence, GLUD2 adaptation may have influenced the evolutionary course taken by the human brain, but understanding the mechanism(s) involved remains challenging.

In Silico Discovery of Plant-Origin Natural Product Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)

An in silico drug discovery pipeline for the virtual screening of plant-origin natural products (NPs) was developed to explore new direct inhibitors of TNF and its close relative receptor activator of nuclear factor kappa-B ligand (RANKL), both representing attractive therapeutic targets for many chronic inflammatory conditions. Direct TNF inhibition through identification of potent small molecules is a highly desired goal; however, it is often hampered by severe limitations. Our approach yielded a priority list of 15 NPs as potential direct TNF inhibitors that were subsequently tested in vitro against TNF and RANKL. We thus identified two potent direct inhibitors of TNF function with low micromolar IC50 values and minimal toxicity even at high concentrations. Most importantly, one of them (A11) was proved to be a dual inhibitor of both TNF and RANKL. Extended molecular dynamics simulations with the fully automated EnalosMD suite rationalized the mode of action of the compounds at the molecular level. To our knowledge, these compounds constitute the first NP TNF inhibitors, one of which being the first NP small-molecule dual inhibitor of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.