RESUMO
One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aß) protein in amyloid plaques in the brain. The Aß peptide exists in several forms, including full-length Aß1-42 and Aß1-40 - and the N-truncated species, pyroglutamate Aß3-42 and Aß4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aß species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aß3-14, we identified a novel pseudo ß-hairpin structure in the N-terminal region of Aß and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aß1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aß, which offers new routes for active and passive immunisation against AD.
Assuntos
Doença de Alzheimer , Vacinas , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos/metabolismo , Encéfalo/metabolismo , Camundongos , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Vacinas/metabolismoRESUMO
The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.
Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Ubiquitina , Ubiquitinação , Regulação Alostérica , Células HEK293 , Humanos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Autophagy is a cell-protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However, there are currently no specific inhibitors of autophagy. ULK1, a serine/threonine protein kinase, is essential for the initial stages of autophagy, and here we report that two compounds, MRT67307 and MRT68921, potently inhibit ULK1 and ULK2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant, we show that the autophagy-inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in accumulation of stalled early autophagosomal structures, indicating a role for ULK1 in the maturation of autophagosomes as well as initiation.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , CamundongosRESUMO
BACKGROUND AND PURPOSE: An ever-growing wealth of information on current drugs and their pharmacological effects is available from online databases. As our understanding of systems biology increases, we have the opportunity to predict, model and quantify how drug combinations can be introduced that outperform conventional single-drug therapies. Here, we explore the feasibility of such systems pharmacology approaches with an analysis of the mevalonate branch of the cholesterol biosynthesis pathway. EXPERIMENTAL APPROACH: Using open online resources, we assembled a computational model of the mevalonate pathway and compiled a set of inhibitors directed against targets in this pathway. We used computational optimization to identify combination and dose options that show not only maximal efficacy of inhibition on the cholesterol producing branch but also minimal impact on the geranylation branch, known to mediate the side effects of pharmaceutical treatment. KEY RESULTS: We describe serious impediments to systems pharmacology studies arising from limitations in the data, incomplete coverage and inconsistent reporting. By curating a more complete dataset, we demonstrate the utility of computational optimization for identifying multi-drug treatments with high efficacy and minimal off-target effects. CONCLUSION AND IMPLICATIONS: We suggest solutions that facilitate systems pharmacology studies, based on the introduction of standards for data capture that increase the power of experimental data. We propose a systems pharmacology workflow for the refinement of data and the generation of future therapeutic hypotheses.
Assuntos
Anticolesterolemiantes/farmacologia , Colesterol/biossíntese , Modelos Biológicos , Biologia de Sistemas , Animais , Anticolesterolemiantes/administração & dosagem , Anticolesterolemiantes/efeitos adversos , Biologia Computacional , Desenho de Fármacos , Quimioterapia Combinada , Humanos , Ácido Mevalônico/metabolismoRESUMO
N-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa Trypanosoma brucei (T. brucei), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (1) as a starting point for the design of a class of potent, brain penetrant inhibitors of T. brucei NMT.
Assuntos
Aciltransferases/antagonistas & inibidores , Aminopiridinas/química , Aminopiridinas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Aciltransferases/metabolismo , Aminopiridinas/síntese química , Aminopiridinas/farmacocinética , Animais , Encéfalo/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Tripanossomicidas/síntese química , Tripanossomicidas/farmacocinética , Tripanossomíase Africana/metabolismoRESUMO
The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Aurora Quinase A/química , Sítios de Ligação/efeitos dos fármacos , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Ligantes , Proteínas Associadas aos Microtúbulos/química , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Ligação Proteica/efeitos dos fármacos , Tiazolidinas/química , Tiazolidinas/farmacologiaRESUMO
New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high-throughput screen of a focussed kinase set of ~3400 compounds to identify potent and parasite-selective inhibitors of an enzymatic Leishmania CRK3-cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3-CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low-nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.