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Copenhagen, Denmark

Mahalka A.K.,Aalto University | Kirkegaard T.,Danish Cancer Society | Kirkegaard T.,Orphazyme | Jukola L.T.I.,Aalto University | And 2 more authors.
Biochimica et Biophysica Acta - Biomembranes

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-δSBD and Hsp70-δNBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD-substrate interaction also within the hydrophobic bilayer interior acyl chain region. © 2014 Elsevier B.V. Source

Kirkegaard T.,Orphazyme
Expert Opinion on Orphan Drugs

Introduction: The success of the first enzyme replacement therapy (ERT) for a lysosomal storage disease (LSD) and the regulatory and commercial incentives provided by authorities for orphan and rare diseases has spawned a massive interest for developing drugs for these intriguing but devastating genetic disorders. The potential for new drugs in this arena is vast, as not only a high number of LSDs have no available therapy, but also alternative therapeutic approaches for diseases with existing treatment are much needed as a number of challenges facing the existing therapies have become very obvious. A significant unmet medical need is therefore apparent for most, if not all of the LSDs and the development of new therapies based on the increasing knowledge of the pathophysiological mechanisms involved in these devastating diseases is therefore anticipated with great interest from all stakeholders. Areas covered: The reader will be introduced to the intricate biological processes involved in lysosomal regulation and how these are exploited for current and emerging therapies. Therapies utilizing these processes will be thoroughly reviewed with regard to their mechanism of action, their clinical status and the challenges they are faced with and/or are aiming to address. For this review, a literature research has been undertaken that covers the years 1955-2012. Expert opinion: The interest in lysosomal biology and disease has surged over the past decade not only in the halls of science but also of pharmaceutical companies. As the complexity of the LSDs increasingly become revealed, so do novel therapeutic targets continuously nurturing the development of new candidate drugs for these devastating diseases. Among this multitude of approaches, the ERTs still account for the vast majority of approved therapies but a number of exciting alternative approaches are emerging targeting various components of the pathophysiological cascade. This evolution of the field is much needed as the presently available treatments are unable to address all clinical aspects of these multifaceted diseases. Future therapy will most likely consist of combinations of these established and emerging approaches as well as other yet to be discovered concepts as the complexity of the diseases demands a certain degree of humbleness to the expectations for a cure based on a single therapy. © Informa UK, Ltd. Source

Ellegaard A.-M.,Danish Cancer Society | Groth-Pedersen L.,Danish Cancer Society | Groth-Pedersen L.,Copenhagen University | Oorschot V.,University Utrecht | And 5 more authors.
Molecular Cancer Therapeutics

Defective apoptosis signaling and multidrug resistance are major barriers for successful cancer treatment. To identify drugs capable of targeting treatment-resistant cancer cells, we screened small-molecule kinase inhibitor libraries for compounds that decrease the viability of apoptosis-resistant human MCF7-Bcl-2 breast cancer cells. SU11652, a multitargeting receptor tyrosine kinase inhibitor, emerged as the most potent compound in the screen. In addition to MCF7-Bcl-2 cells, it effectively killed HeLa cervix carcinoma, U-2- OS osteosarcoma, Du145 prostate carcinoma, and WEHI-S fibrosarcoma cells at low micromolar concentration. SU11652 accumulated rapidly in lysosomes and disturbed their pH regulation and ultrastructure, eventually leading to the leakage of lysosomal proteases into the cytosol. Lysosomal destabilization was preceded by an early inhibition of acid sphingomyelinase, a lysosomal lipase that promotes lysosomal membrane stability. Accordingly, Hsp70, which supports cancer cell survival by increasing lysosomal acid sphingomyelinase activity, conferred partial protection against SU11652-induced cytotoxicity. Remarkably, SU11652 killed multidrug-resistant Du145 prostate cancer cells as effectively as the drug-sensitive parental cells, and subtoxic concentrations of SU11652 effectively inhibited multidrug-resistant phenotype in Du145 prostate cancer cells. Notably, sunitinib, a structurally almost identical and widely used antiangiogenic cancer drug, exhibited similar lysosome-dependent cytotoxic activity, albeit with significantly lower efficacy. The significantly stronger lysosome-targeting activity of SU11652 suggests that it may display better efficacy in cancer treatment than sunitinib, encouraging further evaluation of its anticancer activity in vivo. Furthermore, our data provide a rationale for novel approaches to target drug-resistant cancers by combining classic chemotherapy with sunitinib or SU11652. © 2013 AACR. Source

The invention relates to the use of a chemical substance selected from the group consisting of N-2-hydroxy-3-(1piperidinyl)-propoxy]-1-pyridine-1-oxide-3-carboximidoyl chloride, the optically active enantiomers and the mixtures of enantiomers thereof and pharmaceutically acceptable salts of the racemic and optically active compounds in the preparation of a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases.

Orphazyme | Date: 2014-07-14

The present invention concerns a method for modulating the enzymatic activity of an enzyme, wherein said enzyme interacts with BMP, said method comprising the step of administering or inducing Hsp70, or a functional fragment or variant thereof, in a form suitable for allowing interaction between BMP and Hsp70, or said functional fragment or variant thereof, and thereby modulating the enzymatic activity of an enzyme interacting with BMP.

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