Infectious Disorders - Drug Targets (Formerly Current Drug Targets - Infectious Disorders) - Volume 9, Issue 1, 2009

Prion diseases are strictly fatal neurodegenerative and infectious disorders in man and animal for which no effective therapeutic or prophylactic regimens are presently available. The underlying molecular mechanism is the conformational conversion of the normal cellular prion protein (PrPc) into the pathological isoform PrPSc, accompanied by dramatic changes in biochemical properties. Prion diseases are prototypic for conformational disorders, but are unique in its infectious character and still enigmatic in many aspects. The manifestation of human prion diseases can be acquired by infection, or endogenously as sporadic or genetic disease. Intra- and also inter-species transmission is frequent in animal diseases, with the risk of zoonotic diseases like recently seen with vCJD caused by BSE. vCJD seems rather effectively transmissible within the human population by blood products, resulting in iatrogenic secondary vCJD. The disease itself is restricted to the central nervous system and therefore difficult to diagnose pre-clinically. As prions propagate by conversion of a host-encoded protein, an immune reaction in the host is completely absent and serological markers are missing. This self-tolerance hampers classical prophylactic approaches, e.g. in form of vaccines. Very challenging is also treatment of prion diseases, which asks for compounds able to effectively cross the blood-brain-barrier. The non-availability of means for pre-clinical diagnosis results in advanced stages of brain damage when clinical symptoms become evident. Nevertheless, in animal models a very impressive reversion of pathology and clinical symptoms could be achieved. Although human forms of disease are rare, with an incidence of one in a million world-wide for sporadic CJD, and animal diseases do not really ask for therapy, there is an urgent need for postexposure, therapeutic and eventually prophylactic intervention possibilities, in particular with regard to vCJD and the fact that this is a strictly deadly disease. In this article series, recent advances in therapeutic developments, novel molecular targets, and future perspectives are discussed. Obvious target groups are PrPc and PrPSc themselves, required as functional substrate or template, respectively, for prion conversion. Many intervention strategies address the interaction and the subsequent conversion process of isoforms. Other targets are the cellular clearance capacity for PrPSc, the involvement of putative co-factors, and the neurotoxic effects exerted by yet to characterize toxic PrP species. Finally, experimental proof-of-principle exists for active and passive vaccination strategies. Although various promising drug candidates with anti-prion activity have been identified, this solid proof-of-concept could not yet be transferred into translational medicine.

Prion diseases are infectious and fatal neurodegenerative disorders of man and animals which are characterized by spongiform degeneration in the central nervous system. In human diseases, the manifestation can be sporadic, familial or acquired by infection. Prion disorders are caused by the accumulation of an aberrantly folded isoform of the cellular prion protein (PrPc), commonly named PrPSc. Although prion diseases are usually rare, they have the potential to be transferred within and also between species by infection processes, giving then raise even to epidemic scenarios. As pathology is obviously restricted to the central nervous system pre-mortem diagnosis is usually hard to achieve. Promising approaches towards the development of therapeutic and even prophylactic anti-prion regimens were recently made. However, only a profound knowledge of the infectious agent and its replication strategy enables the design of effective anti-prion strategies. Cell culture models were highly instrumental in uncovering fundamental aspects of prion propagation. In this chapter, the cellular and molecular biology of prion proteins in general is discussed and prophylactic and therapeutic concepts derived thereof are introduced. In particular, emphasis is put on strategies targeting PrPc which is absolutely needed as substrate for prion conversion, and on intrinsic cellular clearance mechanisms for prions.

Recent outbreaks of variant Creutzfeldt-Jakob disease and iatrogenic Creutzfeldt-Jakob disease have aroused great concern in many countries and have necessitated the development of suitable therapies. We have demonstrated that sulfated glycans such as pentosan polysulfate and fucoidan, and amyloidophilic compounds such as amyloid dye derivatives, styrylbenzoazole derivatives, and phenylhydrazine derivatives have efficacies in prion-infected animals. Amyloidophilic compounds present potentialities not only as therapeutic candidates but also as prion amyloid imaging probes for use in nuclear medicine technology such as positron emission tomography. A representative of styrylbenzoazole compounds has been used recently for clinical trials of brain prion amyloid imaging in patients. On the other hand, a representative of phenylhydrazine compounds, compB, displays excellent effectiveness in prolonging the incubation times of infected animals when given orally. However, both its anti-prion effectiveness in vitro and its therapeutic efficacy in vivo are consistently dependent on the prion strain. This prion-strain-dependency is similarly observed in other amyloidophilic compounds. Therefore, aside from further improvement of the safety profiles and pharmacokinetic properties of such compounds, elucidation and management in the mechanism of the prion straindependent effectiveness is necessary. Nevertheless, because compB studies suggest that amyloidophilic compounds are also therapeutic candidates for Alzheimer's disease, amyloidophilic compounds might be attractive as drug candidates for various conformational diseases and hasten development of therapeutic drugs for prion diseases.

In the last decade information has accumulated on the potential anti-prion activity of polycyclic compounds. Initially we showed that the antitumoral idodoxorubicin reduced the infectivity in experimental scrapie. On the basis of the chemical homology with anthracyclines, we rapidly moved to tetracyclines, compounds that are safer and widely used as antibiotics in clinical practice. The tetracyclines, essentially doxycycline and minocycline, were characterized as a therapeutical tool in transmissible spongiform encephalopathies (TSE) through the cell-free condition, in cellular and animal models and they are now being investigated clinically with this indication. Tetracyclines interact with aggregates obtained by synthetic PrP peptides or pathological PrP (PrPsc) extracted from TSE brains, and they destabilize the structure of amyloid fibrils, reducing their resistance to digestion by proteinase K. Tetracyclines also interact with peptide oligomeric structures and inhibit the protein misfolding associated with PrPsc formation. These activities have been accompanied by a reduction of infectivity, verified by doxycycline treatment in experimental scrapie, and some curative effects after either peripheral or intracerebral infection. The anti-amyloidogenic activity of tetracyclines was tested in other forms of peripheral and central amyloidosis, with interesting results. This article analyzes the development of tetracyclines as a therapeutic tool in TSE in the light of recent results obtained in our laboratories.

Prion diseases are fatal and incurable infectious neurodegenerative disorders affecting humans and other mammals. Prions are composed essentially if not solely of PrPSc, a misfolded form of the host-encoded PrP protein. PrPSc catalyzes the transconformation of the normal endogenous PrP (PrPC) into more PrPSc. Prion replication thus corresponds to the propagation of an altered folding state of PrP. Several prion proteins have also been identified in the simple model organism Saccharomyces cerevisiae. Yeast prion-based screening assays have allowed identification of drugs active against mammalian prions, thus revealing the existence of common prion propagation mechanisms conserved from yeast to human. To identify these conserved targets, antiprion compounds isolated in yeast can be used as baits in reverse screening strategies. Once identified, these targets could in turn lead to the development of mechanism-based cell-free antiprion screening assays. A reverse screening procedure has been performed for 6AP and GA, two antiprion compounds isolated using a yeast-based assay. Protein folding activity of the large ribosomal RNA was found to be a physical and a functional target of both 6AP and GA therefore suggesting that this activity of the ribosome may constitute a novel mechanism involved in prion propagation and, as a consequence, a new screening target.

Until now it is still not clear which structural elements of the prion protein (PrP) are involved in its conversion process. Characterisation of these essential regions would help to understand the conversion process itself and might help to develop specific therapeutic approaches to inhibit PrPres formation by dominant inhibitory mutations. To address this important question 33 evenly spaced insertion mutants were generated spanning the entire sequence of the murine 3F4- tagged PrP. The mutants were expressed by retroviral transduction in three different scrapie infected cell lines (ScN2a; SMB[RC040]; SMB[22F]). The convertibility was affected not only by introducing the insertion in the putatively refolded region (aa100-170), but also in the C-terminus of PrP (up to aa214). Moreover, dominant inhibitory effects on conversion were observed for PrP-mutants at four distinguished regions (aa100-112; aa130-154; aa166-172, aa196-200). Computer based structural analysis revealed that these segments were organized in two structurally clearly separated regions supporting the idea that they could function as protein-protein interaction sites which are necessary during seed formation.

Prions are unique in that the infectious particles contain no detectable nucleic acid and appear to consist of aggregated forms of misfolded cellular prion protein. Prions form distinct strains and can transmit disease between species. Whilst the molecular basis of prion diseases is beginning to be unravelled, much remains unknown including the atomic structure of the infectious and toxic species. In contrast, the structure and folding properties of the cellular prion protein are well characterised and, although its precise function remains enigmatic, constitutive knockout of protein expression in mice produces apparently healthy animals but which are fully resistant to prion infection. Furthermore, recent data show that neuronal knockout of the gene encoding for prion protein during established brain infection leads to reversal of pathology and behavioural deficits, giving hope that effective therapies could be designed. Stabilising the cellular form of the prion protein and preventing it from misfolding could be one way to slow or prevent prion formation. Immunotherapy of peripherally prion-infected mice with an antibody specific for cellular prion protein can prevent disease onset. However, a small molecule capable of curing prion infection in vivo has still to be discovered. Recent work has provided proof of principle that compounds which bind selectively to the cellular prion protein could act as therapeutics for prion diseases.

Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal prion protein named PrPSc. PrPSc results from the post-translational conformational modification of the host-encoded protein PrPC. To date there is no treatment for this inexorably fatal disease. Hence, a major focus of research consists in the identification of new molecules that could interfere with in vivo prion propagation. Promising therapeutic approaches to block the production of PrPSc are based on PrP RNA interference, passive or active immunization, dominant negative inhibition of PrPSc formation, as well as inhibition of interactions between PrPSc and other cofactors. Although these anti-prion molecules can be directly administered in vivo, the process to produce and purify them in high quantity is often challenging and expensive. An alternative strategy consists in the development of gene therapy systems of delivery. Importantly, the diagnosis of prion disease in humans remains difficult and often leaves a short therapeutic window after the appearance of the first clinical signs. As serious damages to the brain generally occur before clinical symptoms manifest, an ideal therapeutic strategy must target not only the formation of toxic aggregates, but also the brain destruction already incurred. This could be achieved by combining gene therapy with cell therapy. In this review we have chosen to highlight the multiple targets and potential gene or cell replacement therapeutic approaches. This review also presents the evidence for the transplantation of stem cells as well as the combination of cell and gene therapy as promising strategies against prion diseases.

The 37 kDa/67 kDa laminin receptor (LRP/LR) represents a key player for cell adhesion, is associated with the metastatic potential of solid tumors and is required for maintenance of cell viability by preventing apoptosis. LRP/LR acts as a receptor for viruses such as Sindbis virus, Venezuelean Equine Encephalitis (VEE) virus, Adeno-associated-viruses (AAV) and Dengue Virus, the latter causing 50 to 100 million infections in humans per year. LRP/LR acts further as a receptor for prions and represents a multifunctional protein subcellularly located to the nucleus, the cytoplasm and the cell surface. The receptor represents an alternative target for therapy of viral infections, cancer and prion disorders and might play additional roles in further neurodegenerative diseases such as Alzheimer's disease. The species barrier in prion disorders might be at least in part determined by the presence of LRP/LR in enterocytes of the intestinal epithelium. Anti- LRP/LR antibodies, siRNAs directed against LRP mRNA, polysulfated glycanes such as pentosan polysulfate and heparan mimetics and LRP decoy mutants are promising tools for blocking or downregulating the receptor and may represent alternative therapeutics for the treatment of prion disorders, Alzheimer's Disease and metastatic cancer.

The transmissible spongiform encephalopathies are rapidly progressive and invariably fatal neurodegenerative diseases for which there are no proven efficacious treatments. Many approaches have been undertaken to find ways to prevent, halt, or reverse these prion diseases, with limited success to date. However, as both our understanding of pathogenesis and our ability to detect early disease increases, so do our potential therapeutic targets and our chances of finding effective drugs. There is increasing pressure to find effective decontaminants for blood supplies, as variant Creutzfeldt Jakob Disease (vCJD) has been shown to be transmissible by blood, and to find non-toxic preventative therapies, with ongoing cases of Bovine Spongiform Encephalopathy (BSE) and the spread of Chronic Wasting Disease (CWD). Within the realm of chemotherapeutic approaches, much research has focussed on blocking the conversion of the normal form of prion protein (PrPc) to its abnormal counterpart (PrPres). Structurally, these chemotherapeutic agents are often polyanionic or polycyclic and may directly bind PrPc or PrPres, or act by redistributing, sequestering, or downregulating PrPc, thus preventing its conversion. There are also some polycationic compounds which proport to enhance the clearance of PrPres. Other targets include accessory molecules such as the laminin receptor precursor which influences conversion, or cell signalling molecules which may be required for pathogenesis. Of recent interest are the possible neuroprotective effects of some drugs. Importantly, there is evidence that combining compounds may provide synergistic responses. This review provides an update on current testing methods, therapeutic targets, and promising candidates for chemical-based therapy.

The progress in understanding disease pathology and phenomenology in prion disorders and recent advances in diagnostic techniques might allow researchers to think about therapeutic trials in CJD patients. Some attempts have been made in the past. Drugs tested involved a variety of compounds, which belong to antimicrobial, antiinflammatory or analgesic substance classes. Most papers on this subject describe single case reports. Controlled trials are virtually not available and a double-blinded study was published for flupirtine only. Despite it has been demonstrated that a clinical trial is feasible, the clinicians might face several specific problems when evaluating the efficacy of the drug in CJD, such as rareness of the disease, lack of appropriate preclinical test and heterogeneous clinical presentation in humans. These problems have to be carefully addressed in future trials.