ABSTRACT
Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aß) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.
Subject(s)
Alzheimer Disease/drug therapy , Drug Discovery , Molecular Chaperones/pharmacology , Prealbumin/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Alzheimer Disease/metabolism , Calorimetry , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Chaperones/chemistry , Molecular Structure , Prealbumin/metabolism , Small Molecule Libraries/chemistry , Software , Structure-Activity RelationshipABSTRACT
Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.
Subject(s)
Benzbromarone/chemistry , Drug Repositioning , Neuroprotective Agents/chemistry , Prealbumin/chemistry , Thyroxine/chemistry , Amyloid/antagonists & inhibitors , Benzbromarone/metabolism , Benzoxazoles/chemistry , Benzoxazoles/metabolism , Binding Sites , Binding, Competitive , Crystallography, X-Ray , Diflunisal/analogs & derivatives , Diflunisal/chemistry , Diflunisal/metabolism , Gene Expression , Humans , Hydrogen Bonding , Kinetics , Molecular Docking Simulation , Neuroprotective Agents/metabolism , Prealbumin/agonists , Prealbumin/genetics , Prealbumin/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics , Thyroxine/metabolism , Tolcapone/chemistry , Tolcapone/metabolismABSTRACT
Hepatic adenomatosis is defined as the presence of 10 or more adenomas in an otherwise normal liver. Half of the cases are clinically silent and detected incidentally in imaging exams. A 42-year-old woman with previous history of arterial hypertension and mixed dyslipidemia had multiple liver nodules incidentally identified in an abdominal computed tomography scan. She was asymptomatic and her physical examination was unremarkable but laboratory analysis revealed increased alkaline phosphatase and mildly persistent elevated systemic inflammatory markers. A subsequent hepatic magnetic resonance imaging (MRI) suggested the diagnosis of hepatic adenomatosis and the liver biopsy confirmed the presence of inflammatory adenomas. The patient stopped oral contraception and, at 6 months of follow-up, laboratory inflammatory markers had normalized. She is now under biannual follow-up with MRI and alpha-fetoprotein dosing. This case provides an example of the complex management of this disease in terms of diagnosis, treatment, and follow-up.
A adenomatose hepática define-se pela presença de 10 ou mais adenomas num fígado normal. Cerca de metade dos casos são silenciosos e de identificação incidental em exames de imagem. Uma mulher de 42 anos, com antecedentes pessoais de hipertensão arterial e dislipidémia mista, realizou uma tomografia computorizada abdominal, com identificação incidental de múltiplos nódulos hepáticos. Apresentava-se assintomática e sem alterações ao exame objetivo mas as análises laboratoriais revelaram fosfatase alcalina elevada e aumento ligeiro mas persistente dos parâmetros inflamatórios sistémicos. A ressonância magnética hepática (RMH) subsequente sugeriu o diagnóstico de adenomatose hepática e a biópsia de nódulo confirmou a presença de adenoma do tipo inflamatório. Foi suspensa a toma de contraceptivo oral e, aos 6 meses de follow-up, observou-se normalização dos parâmetros inflamatorios. Actualmente encontra-se sob vigilância semestral com RMH e doseamento de alfa-feto-proteína. Este caso representa a complexidade inerente à abordagem do diagnòstico, tratamento e follow-up da adenomatose hepática.
