Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry

Chaperonas farmacológicas en fenilcetonuria: estudio del desplegamiento de la enzima PAH e identificación de compuestos con propiedades farmacológicas

La fenilcetonuria (PKU) es una enfermedad rara congénita del metabolismo del aminoácido Phe que tiene una herencia autosómica recesiva y está causada por mutaciones en el gen que codifica la enzima humana fenilalanina hidroxilasa (PAH). Estas mutaciones de la proteína provocan una actividad deficiente de la enzima PAH dando lugar a la acumulación de Phe en el organismo a niveles neurotóxicos. La detección precoz de la enfermedad y el seguimiento de una dieta estricta pobre en Phe previenen los daños más graves de la enfermedad. A pesar del éxito de estas medidas, en algunos pacientes se observa una baja adherencia al régimen dietético prescrito debido a las importantes cargas sociales y económicas que conlleva y se han presentado déficits neurocognitivos. La necesidad de nuevas estrategias terapéuticas que incidan en una mejora en la calidad de vida de los pacientes ha motivado numerosas investigaciones a lo largo de estas décadas fructificando en dos nuevas terapias disponibles para pacientes de PKU: el fármaco Kuvan® (formulación sintética del cofactor) o las inyecciones con PEG-PAL. Ninguno de estos tratamientos son útiles o seguros para todos los pacientes PKU, por lo que se siguen estudiando nuevas aproximaciones que complementen o sustituyan las terapias ya existentes. Entre ellas, una de las aproximaciones más prometedoras es el uso de chaperonas farmacológicas. Éstas son compuestos químicos de bajo peso molecular que se unen de manera específica al estado nativo de la proteína inestable, permitiendo la estabilización de la proteína y, por tanto, recuperando su función fisiológica. Bajo este contexto, se ha desarrollado el trabajo de esta tesis doctoral. En ella, se recogen los resultados del estudio del desplegamiento de la enzima PAH monitorizado mediante distintas técnicas biofísicas y de la identificación y la evaluación de una serie de compuestos con potencial como futuras chaperonas farmacológicas. La enzima PAH tiene varios dominios estructurales y se encuentra como homotetrámero en su forma oligomérica funcional. En la actual tesis, se ha estudiado el desplegamiento químico y térmico de la forma completa de la enzima y una forma doblemente truncada de la misma. Los intermediarios de desplegamiento poblados en ambos desplegamientos parecen ser diferentes. Además, el estudio del desplegamiento térmico permite establecer una recomendación útil a la hora de seleccionar compuestos en cribados masivos experimentales basados en ensayos de fluorimetría diferencial de barrido.Por otro lado, se han identificado nuevas moléculas con efecto termoestabilizador in vitro de PAH mediante tres metodologías distintas: diseño racional de compuestos de segunda generación de una chaperona previamente identificada en el grupo, cribado masivo experimental y reposicionamiento de fármacos. Además, se ha evaluado la toxicidad y el efecto rescatador de la actividad enzimática PAH de los compuestos en células. Algunos compuestos identificados muestran un notable efecto rescatador de la actividad enzimática en las distintas variantes patológicas de PAH evaluadas en células. Estos compuestos podrían presentar un comportamiento de chaperona farmacológica canónica o actuar, además, mediante otros mecanismos de acción. El efecto celular de los compuestos en la actividad de PAH difiere del mostrado por el cofactor e indica que es posible personalizar el tratamiento farmacológico en función de la variante PKU expresada. Estos compuestos, previo estudio in vivo de eficacia y toxicidad en modelos animales de la enfermedad, podrían ofrecer nuevas posibilidades terapéuticas a los pacientes de esta enfermedad que complementen o sustituyan las terapias existentes. <br /

A pyrene-inhibitor fluorescent probe with large stokes shift for the staining of Aβ1–42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing staphylococcus aureus biofilms

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them.IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SVis supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government

ZPD-2, a small compound that inhibits α-synuclein amyloid aggregation and its seeded polymerization

Altres ajuts: SV was supported by the ICREA (ICREA-Academia 2015) and the Fundación La Marató de TV3 (Ref. 20144330). JavS was supported by the Gobierno de Aragón (E45_17R). ED was supported by the Instituto de Salud Carlos III (PH613883/ERDF/ESF).α-Synuclein (α-Syn) forms toxic intracellular protein inclusions and transmissible amyloid structures in Parkinson's disease (PD). Preventing α-Syn self-assembly has become one of the most promising approaches in the search for disease-modifying treatments for this neurodegenerative disorder. Here, we describe the capacity of a small molecule (ZPD-2), identified after a high-throughput screening, to inhibit α-Syn aggregation. ZPD-2 inhibits the aggregation of wild-type α-Syn and the A30P and H50Q familial variants in vitro at substoichiometric compound:protein ratios. In addition, the molecule prevents the spreading of α-Syn seeds in protein misfolding cyclic amplification assays. ZPD-2 is active against different α-Syn strains and blocks their seeded polymerization. Treating with ZPD-2 two different PD Caenorhabditis elegans models that express α-Syn either in muscle or in dopaminergic (DA) neurons substantially reduces the number of α-Syn inclusions and decreases synuclein-induced DA neurons degeneration. Overall, ZPD-2 is a hit compound worth to be explored in order to develop lead molecules for therapeutic intervention in PD

Multiple myeloma and SARS-CoV-2 infection: clinical characteristics and prognostic factors of inpatient mortality

There is limited information on the characteristics, prognostic factors, and outcomes of patients with multiplemyeloma (MM) hospitalized with COVID-19. This retrospective case series investigated 167 patients reported from 73hospitals within the Spanish Myeloma Collaborative Group network in March and April, 2020. Outcomes werecompared with 167 randomly selected, contemporary, age-/sex-matched noncancer patients with COVID-19 admittedat six participating hospitals. Among MM and noncancer patients, median age was 71 years, and 57% of patients weremale; 75 and 77% of patients, respectively, had at least one comorbidity. COVID-19 clinical severity wasmoderate-severe in 77 and 89% of patients and critical in 8 and 4%, respectively. Supplemental oxygen was requiredby 47 and 55% of MM and noncancer patients, respectively, and 21%/9% vs 8%/6% required noninvasive/invasiveventilation. Inpatient mortality was 34 and 23% in MM and noncancer patients, respectively. Among MM patients,inpatient mortality was 41% in males, 42% in patients aged >65 years, 49% in patients with active/progressive MM athospitalization, and 59% in patients with comorbid renal disease at hospitalization, which were independentprognostic factors on adjusted multivariate analysis. This case series demonstrates the increased risk and identifiespredictors of inpatient mortality among MM patients hospitalized with COVID-19

Multiple myeloma and SARS-CoV-2 infection: clinical characteristics and prognostic factors of inpatient mortality

There is limited information on the characteristics, prognostic factors, and outcomes of patients with multiple myeloma (MM) hospitalized with COVID-19. This retrospective case series investigated 167 patients reported from 73 hospitals within the Spanish Myeloma Collaborative Group network in March and April, 2020. Outcomes were compared with 167 randomly selected, contemporary, age-/sex-matched noncancer patients with COVID-19 admitted at six participating hospitals. Among MM and noncancer patients, median age was 71 years, and 57% of patients were male; 75 and 77% of patients, respectively, had at least one comorbidity. COVID-19 clinical severity was moderate–severe in 77 and 89% of patients and critical in 8 and 4%, respectively. Supplemental oxygen was required by 47 and 55% of MM and noncancer patients, respectively, and 21%/9% vs 8%/6% required noninvasive/invasive ventilation. Inpatient mortality was 34 and 23% in MM and noncancer patients, respectively. Among MM patients, inpatient mortality was 41% in males, 42% in patients aged >65 years, 49% in patients with active/progressive MM at hospitalization, and 59% in patients with comorbid renal disease at hospitalization, which were independent prognostic factors on adjusted multivariate analysis. This case series demonstrates the increased risk and identifies predictors of inpatient mortality among MM patients hospitalized with COVID-19

Identification and development of new pharmacological chaperones for phenylketonuria

Resumen del póster presentado a la VII National Conference of the Institute for Biocomputation and Physics of Complex Systems (BIFI), celebrada en Zaragoza (España) del 4 al 6 de febrero de 2015.Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutations of phenylalanine hydroxylase (PAH) gene. Nowadays, more than 550 disease-causing PAH mutations have been identified and most of them induce loss of conformational stability and decreased physiological enzymatic activity. Currently, one of the most promising molecular approaches to treat this disease involves using small molecules known as pharmacological chaperones that bind to the native state of the unstable enzyme rescuing its physiological function. Actually, BH4, the natural cofactor of PAH, is administered for the management of some PKU patients and it is believed that it acts through that mechanism. Despite this fact, BH4 is only effective for some forms of mild PKU so that we propose to find novel PKU chaperones unrelated to BH4. A previous work of this group identified two such compounds that enhanced the thermal stability of the WT enzyme and solved the X-ray structure of the complex between the enzyme and one of the chaperones. Based on these findings, we are synthetizing a group of novel compounds and testing their effect on thermal denaturation assays. Concurrently, we are testing over 10,000 chemical compounds of a commercial diversity chemical library by applying a high-throughput screening method. These new compounds may represent promising pharmacological entities for non BH4 responsive PKU patients.Peer Reviewe

Studying the mechanism of action of novel stabilizing compounds of phenylalanine hydroxylase

Resumen del póster presentado a la VIII National Congress of the Institute for Biocomputation and Physics of Complex Systems (BIFI), celebrado en Zaragoza (España) del 31 de enero al 2 de febrero de 2017.Peer reviewe

Development of novel stabilizing compounds of human phenylalanine hydroxylase for PKU patients

Resumen del póster presentado al VIII International Congress BIFI: "Complexity, Networks and Collective Behaviour", celebrado en Zaragoza (España) del 6 al 8 de febrero de 2018.Peer reviewe

Synthesis and testing of fluorescent chemical compounds as diagnostic tools for Alzheimer's disease

Resumen del póster presentado a la VII International Conference on Molecular Recognition, celebrada en Zaragoza (España) del 1 al 3 de febrero de 2016.The most common neurodegenerative disease that causes dementia in humans is Alzheimer’s disease. This degeneration of neurons and synapses in the cerebral cortex is characterized by the loss of memory and other very important human skills like reasoning, abstraction and language. Nowadays, around 30 million people are affected by this disease. Histologically, Alzheimer’s disease is characterized by the presence of two microscopic lesions: extracellular senile (amyloid) plaques and neurofibrillary tangles. The senile plaques, also known as neuritic plaques, are made of deposits of amyloid beta peptide (ABeta), which derives from beta amyloid precursor protein (APP) by consecutive proteolytic cleavages. In a previous study, four chemical compounds were found to act as inhibitors of the in vitro and in vivo aggregation of ABeta peptide. In order to develop novel inhibitors that, furthermore, are capable of being used as diagnostic tools in medicine, we have synthesized two compounds derived from one of them in which we have joined a fluorescent dye through two linkers of different length. Our later HTS turbidity tests have shown that this two new compounds keep the inhibitor activity of their predecessor, especially that one in which the linker is longer, what is considered a satisfactory starting point for coming studies.Peer Reviewe