CytRx - Technical Rationale

A New Approach to ALS Pharmacotherapy:

Chaperone Amplification by Arimoclomol

Technical Rationale

CytRx Corporation

November, 2005

Protein misfolding in neurodegenerative diseases

Genetic information stored in DNA manifests itself by acting as a blueprint for the production of proteins. Proteins must assume a specific three dimensional conformation in order to perform their normal biological function. If a protein becomes misfolded, it may no longer be able to perform the biological function for which it was made. Certain proteins can become misfolded in a way that not only prevents it from performing its normal "good" function, but actually causes the protein to gain a "bad" function. Thus, certain misfolded proteins can actually become toxic for the cell in which they reside, resulting in disease^1-3. A common feature of such misfolded toxic proteins is that they tend to "aggregate", that is they stick together to form large clumps of protein.

Protein misfolding has been implicated in the pathogenesis of several neurodegenerative diseases including sporadic and familial amyotrophic lateral sclerosis (ALS and fALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and related polyglutamine (polyQ) expansion diseases, and Transmissible Spongiform encephalopathy (TSE) diseases^4-8. Although the misfolded proteins that aggregate in these disorders are unrelated in size or primary amino acid sequence, the characteristic lesions of each disease are very similar, typically containing fibrillar, amyloid-like structures with common biochemical characteristics such as detergent �insolubility, cross β-structures, protease resistance, and the ability to bind lipophilic dyes such as Congo red. These commonalities indicate that a conserved mechanism of pathogenesis might connect these otherwise phenotypically diverse diseases^4-8.

The original disease-initiating event may be a crucial conformational change that occurs in the disease protein, possibly mediated by physical trauma, oxidative or chemical damage, diet, or an infectious agent ^9-14. Once this initiating event occurs, these misfolded proteins actually have the ability to cause normally folded proteins to misfold^9,15-23, inducing aggregation of the endogenous normal protein. In this sense, misfolded proteins have one of the characteristics of an infectious agent - they can propagate themselves. This may explain the inexorable progressive nature of neurodegenerative diseases. In most instances these aggregated protein products are found to be cytotoxic to neuronal cells in culture. Although the exact mechanism of toxicity is unclear, one possible mechanism of toxicity is the "trapping" of critical proteins that are prone to misfolding. This could explain how protein aggregates initiated by misfolding of different proteins also include other proteins that are common among all aggregates^6,7,24. Post-mitotic cells, such as neurons, may be particularly vulnerable to the detrimental effects of misfolded and/or aggregated proteins because they cannot dilute potentially toxic species through cell division.

There is significant evidence that misfolded protein aggregates can be directly cytotoxic³. However, recent observations indicate that the process of protein aggregation proceeds via several metastable intermediate structures, such as spherical oligomers, protofibrils and pore-like annular structures that are the actual cytotoxic species^6,7,25. The universality of this "toxic oligomer" hypothesis is supported by the finding that a single monoclonal anti�body can recognize a common conformational epitope that is displayed by several otherwise unrelated disease-associated precursor oligomers, including those formed by Aβ, α-synuclein and polyQ-containing pep�tides²⁵. Furthermore, co-incubation of the anti-oligomer antibody with oligomers of the various disease proteins blocks their toxicity when applied to cultured cells, indicating that oligomeric structures formed by distinct disease proteins might confer toxicity through a similar conformational misfolding mechanism.

Fig 1. Summary of proposed involvement of protein misfolding in neurodegenerative diseases

The importance of chaperone proteins in neurodegenerative disease

A highly conserved class of proteins called molecular chaperones has evolved to prevent inappropriate interactions within and between non-native polypeptides, to enhance the effi�ciency of de novo protein folding, and to promote the refolding repair of proteins that have become misfolded as a result of cellular stress^26-28. In addition to this protein repair activity, chaperones can mediate targeting to the proteasome system or to lysosomes, resulting in selective degradation of the misfolded protein when the chaperones cannot repair the misfolded proteins^26-29. These activities of the molecular chaperones may be sufficient to prevent the normal accu�mulation of misfolded proteins. Furthermore, the expression level of these chaperones is increased under conditions of cellular stress, adjusting to the consequent increase in damaged proteins. However, under certain pathological conditions (perhaps due to prolonged stress during chronic disease), the capacity of this protein quality control machinery is apparently exceeded and misfolded proteins then accumulate to dangerous levels. In fact, it is commonly observed in cellular neurodegenerative models that the chaperone and proteasome machinery of the cell become overwhelmed in the presence of pathogenic misfolded proteins^30-33. Thus, improving the capacity of stressed cells to deal with misfolded proteins may prove to be therapeutically beneficial in "protein misfolding diseases" such as neurodegenerative disease.

There is enormous scientific interest in trying to increase the level of intracellular chaperones therapeutically in neurodegenerative diseases^6,24,34,35. There is substantial experimental data that suggests that increased chaperone expression could be beneficial in PD, AD, HD, and several less well known neurodegenerative diseases such as spinocerebellar ataxia (SCA-1 and SCA-3) and spinal and bulbar muscular atrophy (SBMA)^6,24,34,35. With regard to potential therapeutic benefit of chaperones in ALS, intranuclear co-microinjection of expression vectors for the Hsp70 chaperone and mutant SOD1 into primary motor neurons reduces the toxicity of mutant SOD1 (which causes 2% of all ALS cases³⁶), decreases SOD1 aggregation, and enhances cell survival³⁷ compared with the injection of the SOD1 vector alone. Over-expression of Hsp70 in the presence of mutant SOD1 in an N2a (neuroblastoma-like) cell model also ameliorates toxicity³⁸. Injection of exogenous HSP70 in vivo has been shown to inhibit motor neuron degeneration in a neonatal mouse sciatic nerve axotomy model³⁹. The cytoprotective effects of HSP70 seem to be synergistically improved by co-expression of HSP27⁴⁰. This may explain why over-expression of HSP70 alone in transgenic mouse models did not prevent the onset of experimental ALS⁴¹.

Importantly, motor neurons apparently have a high intrinsic threshold for induction of chaperones as a result of cell stress and are thus more sensitive to the toxic effects of protein misfolding^42-44. Thus, increasing the expression of molecular chaperones might prove beneficial for the treatment of neurodegenerative diseases in general, and ALS in particular^45-47.

Arimoclomol for treatment of ALS

Chaperone proteins from spinal tissues are in fact slightly increased in astroglia and motor neurons in response to either physical damage^48-50 or disease induced damage in the ALS mouse model⁵¹. However, in chronic diseases like ALS the unaided cellular stress response seems to be insufficient to cope with prolonged exposure to a stressful environment. In motor neurons, the toxicity of mutant SOD1 protein seems to reduce the availability of chaperones by sequestering them in protein aggregates³³, thereby disrupting their normal chaperone and anti-apop�totic functions and reducing their cytoprotective effects. Strategies aimed at increasing levels of chaperones may therefore be successful in protecting motor neurons from cell death in ALS.

Arimoclomol is a new pharmaceutical that is being developed for the treatment of ALS. The drug is a structurally modified form of bimoclomol (see ref 52 for review), an orally available pharmaceutical previously shown to have broad therapeutic potential in animal models for a wide variety of diseases, all having in common the suspected involvement of misfolded proteins. Bimoclomol amplifies the natural cellular stress response that protects the cell from misfolded proteins by increasing the stress-induced expression of multiple chaperone proteins, including Hsp60, Hsp70, Hsp90 and GRP94, without increasing their expression in the absence of stress⁵². It apparently does this by increasing the DNA binding stability of the transcription factor HSF-1 by increasing its level of phosphorylation⁵³.

Arimoclomol was discovered as a metabolic by-product of bimoclomol. It demonstrated improved therapeutic potential compared to bimoclomol in the treatment of experimental diabetic neuropathy⁵⁴ and also has significant pharmacokinetic advantages, particularly in humans (unpublished). Arimoclomol has successfully undergone extensive toxicological examination in several animal species and has been shown to be well tolerated in two Phase I clinical trials in healthy volunteers (unpublished). Like bimoclomol, arimoclomol amplifies the production of chaperone proteins under conditions of cell stress by prolonging the phosphorylation and consequent activation of HSF-1⁵¹. This is especially important for the protection of motor neurons in ALS since the relatively poor response to misfolded proteins seen in motor neurons is apparently due to a failure to sufficiently activate HSF-1⁴⁴. Thus, arimoclomol improves the repair of misfolded proteins by amplifying the levels of chaperone protein expression, but only in cells that are already pathologically stressed.

The pharmacologic increase of chaperones levels with arimoclomol could potentially protect motor neurons from cell death in ALS. Indeed, previous studies have shown that increasing chaperone levels by treatment with arimoclomol inhibits destruction of spinal motor neurons and consequent muscular atrophy in an in vivo nerve injury model of motor neuron degeneration⁴⁹. Arimoclomol also improved sensory neuron morphological markers and restored functional properties in the sensory system following peripheral nerve injury in rats⁵⁰. Most importantly, treatment with arimo�clomol in a mouse model for ALS prevented neuronal loss, protected muscle function, improved behavioral phenotypes, and extended survival rates by 22%, even when administered after the onset of neuroparalytic symptoms⁵¹. This landmark publication was the first report of a pharmaceutical that could be effective after the onset of symptoms⁵⁵, a necessity for an effective ALS therapy, since human diagnosis occurs substantially after the onset of symptoms in all sporadic ALS. This post-symptomatic efficacy in the ALS mouse model distinguishes it from the other drugs that have previously failed in ALS clinical trials.

The pathological consequences of ALS are numerous. Previous pharmaceutical approaches for ALS intervention⁵⁶ have targeted such diverse aspects of the disease as intracellular oxidation (Vitamin E, Co-enzyme Q), mitochondrial dysfunction (Creatine, TCH346), neurotransmitter transport (Riluzole, topiramate) that consequently causes increased intracellular Ca++ release (verapamil, nimodipine), apoptosis (IGF-1, BDNF, minocycline), and inflammation (cyclosporine, celebrex). If these phenomena are consequences of the disease, not the cause of the disease, this might explain the lack of substantial therapeutic benefit for these classes of pharmaceutics. It is possible that this newly recognized aspect of the disease, protein misfolding, may be the underlying problem that leads to all of these sequelae. Thus, the loss of chaperone protein repair and degradation functions due to protein misfolding may lead to or intensify the effects of oxidative damage that would otherwise be protected by chaperones^57-60. Chaperone inhibition by protein misfolding has also been linked to mitochondrial dysfunction^61,62, perhaps by interfering with the role of chaperones for the normal import of mitochondrial proteins⁶³. Similarly, inhibition of molecular chaperones by protein misfolding has been linked to glutamate excitotoxicity^64-68, apoptosis^69-71, and inflammation^72-74. This may explain why arimoclomol was so effective in several different animal models of acute and progressive neuronal degeneration^49-51.

Combating protein misfolding by amplification of the chaperone response with arimoclomol is an exciting new therapeutic approach for treating ALS. Furthermore, given the apparent underlying feature of protein misfolding in a plethora of other diseases, arimoclomol may show promise as a pharmaceutical intervention for other degenerative diseases like AD, PD, and HD and other apparent diseases of protein misfolding such as Cystic Fibrosis⁷⁶ and Type 2 diabetes⁷⁷.

Fig 2. Super-induction of Protein Chaperones by Arimoclomol Improves Intracellular Elimination of Toxic Damaged Proteins. Toxic proteins arise from conformational changes that occur either as a result of gene mutation or physical and chemical stress. Unlike most damaged proteins that simply lose their biological activity, some proteins can assume a conformation that results in the gain of a toxic function, such as aggregation. These toxic proteins also tend to stimulate the same conformational change in other proteins, acting as catalysts for the generation of more toxic protein. Recognition followed by repair, degradation, or segregation of the offending protein is therefore a critical function to the survival of pathologically stressed cells. Arimoclomol prolongs the activation the active, trimeric, hyper-phosphorylated form of HSF-1, which further activates the transcription of multiple chaperone proteins. Chaperones then refold the toxic protein or, if that is prevented, the protein is tagged by ubiquitin ligases that associate with the protein chaperones. The ubiquitin-tagged protein is then degraded by the proteasome or the lysosome, thereby preventing its aggregation.

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