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Oligomer modulator anle138b and related compounds in neurodegeneration and beyond

dc.contributor.advisorGriesinger, Christian Prof. Dr.
dc.contributor.authorRyazanov, Sergey
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titleOligomer modulator anle138b and related compounds in neurodegeneration and beyondde
dc.contributor.refereeGriesinger, Christian Prof. Dr.
dc.description.abstractengNeurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt-Jakob disease are characterized by a progressive deterioration of brain function as a result of a gradual loss of nerve cells. Due to the fact that age is the dominant risk factor for these diseases and life expectancy is constantly increasing, neurodegenerative disorders are considered as one of the serious future challenges for health care system. To date there is no treatment option capable of slowing, preventing or reversing the progressive death of neurons in brain. Therefore, a development of disease-modifying drugs with neuroprotective and/or neurorestorative properties has a highest priority of pharmaceutical research. In majority of the neurodegenerative disorders, the misfolding and aggregation of specific protein(s) in the brain are the permanent histopathological hallmarks. Among the proposed mechanisms of neurotoxicity, protein misfolding may probably compromise a central cascade that leads to loss of neurons. Based on growing evidence indicating that pathological oligomers rather than large fibrillar deposits constitute the key neurotoxic species, we set out to discover small molecules interfering with pathological oligomerization. The main goal of my thesis was to design, synthesize and assess novel compounds that can modulate in vitro and in vivo the aggregation/misfolding of amyloidogenic proteins, including prion protein, α-synuclein and others. In the initial phase of the project, a high-throughput screening campaign, which was conducted by Prof. Armin Giese, identified a novel class of anti-prion compounds, termed 3,5-DiPhenyl-Pyrazoles. In an attempt to optimize the DPP lead structure and to get insight into the details of structure-activity relationship, we designed and synthesized a focused library of 150 DPP-related compounds. We utilized a fragment-based structure design and an iterative approach that comprised several rounds of structure optimization and synthesis on a basis of in vitro and in vivo testing and SAR analysis. Additionally, we proposed a flexible synthetic scheme allowing straightforward synthesis of the desired compounds. By using a range of in vitro prion protein aggregation assays including SIFT assay, PMCA and RML-infected SMB cell model, we assessed the efficacy of DPP compounds as inhibitors of prion protein misfolding. Our in vitro findings support the pronounced antiprion activity of novel compounds sharing the DPP-based core structure. In PMCA assay and in the cell-based model, compound anle138b shows the highest anti-prion activity against various prion stains, including RML, sCJD and vCJD. On the basis of the results from the SIFT, cell culture and PMCA assay, 38 DPP-related compounds were tested in vivo in RML prion-infected mice model. Following oral administration, anle138b and other DPP compounds strongly inhibited the accumulation of PrPSc, neuronal degeneration and disease progression. Regarding the mode of action of anle138b, several lines of evidences from in vitro and in vivo studies support the mechanism associated with direct modulation of prion protein aggregation at the oligomer level. The second part of my thesis describes the property of selected DPP compounds to interfere with α-synuclein aggregation and toxicity. Here, we conducted the SIFT screening assay on α-synuclein. We were able to identify a set of highly potent inhibitors of α-synuclein aggregation and, moreover, we performed a detailed analysis of structure-activity relationships within our focused library of DPP compounds. Next, the molecular mechanism underlying the modulation of α-synuclein misfolding by DPP compounds was studied by various biochemical and biophysical approaches. By using ThioT aggregation assay, electron microscopy, atomic force microscopy and dynamic light scattering, we showed that water- soluble compounds (sery166a, anle138c, anle145c, sery139 and others) prevent fibrillization of α-synuclein in vitro by stabilization of small globular heterogeneous oligomeric species. CD data suggests that sery166a stabilizes α-synuclein in predominantly unfolded conformation accompanied a low degree of local secondary structure ordering in comparison to the monomeric α-synuclein. To elucidate the mode of inhibition, we applied a high- resolution NMR spectroscopy. HSQC-based NMR binding studies showed a lack of direct binding of all tested compounds to monomeric α-synuclein. By using STD NMR experiments, we demonstrated that the DPP compounds preferentially interact with oligomeric species, while the affinity to monomeric α-synuclein is weak. It’s, therefore, envisioned that the mechanism of aggregation inhibition by DPP compounds might be associated with the interactions between small ligands and oligomeric forms that, in turn, lead to stabilization of specific off-pathway oligomers and, ultimately, blocking of the fibril formation. Considering that the oligomer-induced membrane permeabilization represents a potential toxicity mechanism associated with α-synuclein misfolding, we investigated DPP compounds and compound-stabilized α-synuclein oligomers using electrophysiological studies in planar lipid bilayers. Our analysis of the electrophysiological data reveals that DPP compound-stabilized oligomers do not adversely affect the membrane stability. Remarkably, the addition of DPP compounds either in solution (sery166a, anle138c) or inside the lipid bilayer strongly interferes with membrane permeabilization induced by toxic oligomeric forms of α-synuclein. Finally, the DPP-stabilized α-synuclein oligomers do not alter the magnitude of long-term potentiation (LTP) induced in rat hippocampal slices. In vivo, the water-soluble compound anle145c that efficiently modulates the aggregation of α-synuclein in vitro was found to be active in non-mammalian α-synuclein models. In mammals, anle138b is neuroprotective in several mouse models of synucleinopathies. Of particular significance is the finding that anle138b prevents the formation of toxic oligomers and improves the behavioral, neuropathological and biochemical outcomes in all different animal models based on toxins (Sub-acute MPTP and low-dose intragastric rotenone, the latter being considered also a spreading model), spreading (Lewy body seeding mouse model) and overexpression (Thy1-A30P α-synuclein; 1-120 α- synuclein, PLP-α-synuclein). Our findings provide compelling evidence that anle138b directly modulates the aggregation of α-synuclein at the oligomer level in vivo as shown by sucrose gradient centrifugation assay and super-resolution imaging dSTROM. It also is noteworthy that with prolonged treatment, anle138b has no apparent toxicity in therapeutic doses. In a further part of my thesis I discuss the pharmacokinetic (PK) properties of DPP molecules. Our pharmacokinetic studies have demonstrated that anle138b and other DPP compounds have in general a good oral bioavailability and readily cross the blood-brain- barrier. In case of anle138b, we showed that this molecule has a favorable pharmacokinetic profile in mice and rats. Moreover, we have developed and characterized in PK experiment the special anle138b-containing dry food pellets, which provides convenient way to conduct efficacy studies in mice. Finally, we have identified and tested optimized anle138b formulation V34, which is based on pharmaceutically acceptable excipients. In the last part of my thesis I describe the development of an anle138b prodrug. Due to low aqueous solubility of anle138b, the oral application of this compound requires the dissolution in appropriate excipients. In order to improve the formulation protocol and potentially anle138b’s bioavailability, we designed and synthesized several water-soluble anle138b prodrugs. Among three candidates, the prodrug sery433 showed favorable physicochemical (good stability and high solubility in water) and pharmacokinetic profiles. The comparative pharmacokinetic study in rats with sery433 and anle138b, which were applied orally at the same dose, demonstrated a significant increase of anle138b systemic exposure in the case of prodrug sery433. The mechanism of biotransformation of sery433 in vivo is presumably associated with the enzymatic cleavage of the phosphate moiety by the membrane-bound intestinal phosphatase. Collectively, sery433 is a promising water-soluble anle138b prodrug for further preclinical development. Viewed together, our observations and the results from many other studies strongly support the notion that interference with the protein misfolding by small molecules may represent the attractive strategy in development of disease-modifying therapy. The validity of the aggregation inhibition as a target for drug design was recently corroborated by positive results of the clinical trials of Aducanumab, a monoclonal antibody against a conformational epitope found on misfolded amyloid beta, in early Alzheimer's Disease. Anle138b modulates the oligomer formation of different proteins by targeting structure-dependent epitopes, which are common in various types of pathological aggregates. The efficacy and preclinical data that were collected for the oligomer modulator anle138b strongly supports the selection of this molecule as a candidate for clinical trials in CJD and synucleinopathies. A phase 1 clinical trial with anle138b is scheduled for Q1
dc.contributor.coRefereeDiederichsen, Ulf Prof. Dr.
dc.subject.engprotein aggregationde
dc.subject.engParkinson's diseasede
dc.subject.engprion proteinde
dc.subject.engaggregation inhibitorde
dc.subject.engoligomer modulatorde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

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