Christian Tränkle

Histone deacetylase inhibitors: possible implications for neurodegenerative disorders

During the past six years numerous studies identified histone deacetylase (HDAC) inhibitors as candidate drugs for the treatment of neurodegenerative disorders. Two major neuroprotective mechanisms of HDAC inhibitors have been identified, namely the transcriptional activation of disease-modifying genes and the correction of perturbations in histone acetylation homeostasis, which have been shown to be intimately involved in the neurodegenerative pathomechanisms of Huntingtons, Parkinsons and Kennedy disease, amyotropic lateral sclerosis, Rubinstein-Taybi syndrome as well as stroke. Based on the promising in vitro and in vivo analyses, clinical trials have been initiated to evaluate the safety and efficacy of HDAC inhibitors for the treatment of devastating diseases such as Huntingtons disease, amyotropic lateral sclerosis and spinal muscular atrophy. Here, the authors summarize and discuss the findings on the emerging field of epigenetic therapy strategies in neurodegenerative disorders.

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non‐toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an α‐motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone‐rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m‐Carboxycinnamic acid bis‐Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS‐275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood–brain barrier, oral administration may allow deceleration of progressive α‐motoneurone degeneration by epigenetic SMN2 gene activation.

Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity

Selective modulation of cell function by G protein‐coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype‐selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine‐like orthosteric activator with M2‐selective bis(ammonio)alkane‐type allosteric fragments. Radioligand binding in wild‐type and mutant receptors supplemented by receptor docking simulations proved M2 selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid‐induced dynamic mass redistribution in CHO‐hM2 cells disclosed pathway‐specific signaling. Selective receptor activation (M2>M1>M3) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.— Antony, J., Kellershohn, K., Mohr‐Andrä, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Tränkle, C., Kostenis, E., Christopoulos, A., Höltje, H.‐D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23, 442–450 (2009)

Suberoylanilide hydroxamic acid (SAHA) has potent anti-glioma properties in vitro, ex vivo and in vivo

Current treatment modalities for malignant gliomas do not allow long‐term survival. Here, we identify suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylases (HDAC), as an effective experimental anti‐glioma agent. Administration of SAHA to various glioma cell lines obtained from human, rat and mouse inhibited tumour cell growth in a range of 1–10 μm. This anti‐glioma property is associated with up‐regulation of the cell cycle control protein p21/WAF, as well as the induction of apoptosis. A novel tumour invasion model using slice cultures of rat brain corroborated the anti‐glioma properties of SAHA in the organotypic brain environment. In this model, glioma invasion compromised adjacent brain parenchyma, and this tumour‐associated cytotoxicity could be inhibited by SAHA. In addition, a 10‐fold dose escalation experiment did not challenge the viability of cultured brain slices. In vivo, a single intratumoural injection of SAHA 7 days after orthotopic implantation of glioma cells in syngeneic rats doubled their survival time. These observations identify chromatin‐modifying enzymes as possible and promising targets for the pharmacotherapy of malignant gliomas.

Rational design of dualsteric GPCR ligands: quests and promise

Dualsteric ligands represent a novel mode of targeting G protein‐coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype‐selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR‐complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine‐tuned GPCR‐modulation.

Allosteric ligands for G protein-coupled receptors: A novel strategy with attractive therapeutic opportunities

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter‐bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein‐coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated.

Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275

Inhibitors of histone deacetylases are promising compounds for the treatment of cancer but have not been systematically explored in malignant brain tumors. Here, we characterize the benzamide MS-275, a class I histone deacetylase inhibitor, as potent drug for experimental therapy of glioblastomas. Treatment of four glioma cell lines (U87MG, C6, F98, and SMA-560) with MS-275 significantly reduced cell growth in a concentration-dependent manner (IC90, 3.75 μmol/L). Its antiproliferative effect was corroborated using a bromodeoxyuridine proliferation assay and was mediated by G0-G1 cell cycle arrest (i.e., up-regulation of p21/WAF) and apoptotic cell death. Implantation of enhanced green fluorescent protein–transfected F98 glioma cells into slice cultures of rat brain confirmed the cytostatic effect of MS-275 without neurotoxic damage to the organotypic neuronal environment in a dose escalation up to 20 μmol/L. A single intratumoral injection of MS-275 7 days after orthotopic implantation of glioma cells in syngeneic rats confirmed the chemotherapeutic efficacy of MS-275 in vivo. Furthermore, its propensity to pass the blood-brain barrier and to increase the protein level of acetylated histone H3 in brain tissue identifies MS-275 as a promising candidate drug in the treatment of malignant gliomas. [Mol Cancer Ther 2006;5(5):1248–55]

Extracellular Loop 2 Of The Free Fatty Acid Receptor 2 Mediates Allosterism Of A Phenylacetamide Ago-Allosteric Modulator

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

The peroxisome proliferator-activated receptor-gamma agonist troglitazone inhibits transforming growth factor-beta-mediated glioma cell migration and brain invasion

Gliomas are the most common primary tumors of the central nervous system, with glioblastomas as the most malignant entity. Rapid proliferation and diffuse brain invasion of these tumors are likely to determine the unfavorable prognosis. Considering its promigratory properties, the transforming growth factor-β (TGF-β) signaling pathway has become a major therapeutic target. Analyses of resected glioma tissues revealed an intriguing correlation between tumor grade and the expression of TGF-β1-3 as well as their receptors I and II. Here, we analyzed the effects of peroxisome proliferator-activated receptor γ (PPAR-γ) agonists on glioma proliferation, migration, and brain invasion. Using an organotypic glioma invasion model, we show that micromolar doses of the PPAR-γ activator troglitazone blocked glioma progression without neurotoxic damage to the organotypic neuronal environment observed. This intriguing antiglioma property of troglitazone seems to be only partially based on its moderate cytostatic effects. We identified troglitazone as a potent inhibitor of glioma cell migration and brain invasion, which occurred in a PPAR-γ–independent manner. The antimigratory property of troglitazone was in concordance with the transcriptional repression of TGF-β1-3 and their receptors I and II and associated with reduced TGF-β release. Due to its capacity to counteract TGF-β release and glioma cell motility and invasiveness already at low micromolar doses, troglitazone represents a promising drug for adjuvant therapy of glioma and other highly migratory tumor entities. [Mol Cancer Ther 2007;6(6):1745–54]

Allosteric Small Molecules Unveil a Role of an Extracellular E2/Transmembrane Helix 7 Junction for G Protein-coupled Receptor Activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M2 acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M2Trp422 in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.