Our unique and comprehensive approach leverages industry experience and a nonprofit mission - focused 100% on ALS research.
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We are the world's foremost drug discovery center focused solely on ALS. Pioneering the ‘nonprofit biotech’ model,
we have placed the person with ALS at the center of every decision made in our labs. Our interdisciplinary, industry-trained
team of scientists collaborate daily in our labs and through academic and pharmaceutical company partnerships across the globe
to discover cures for ALS.
To date, ALS TDI has conducted a number of clinical trials. We are currently planning a trial of AT-1501, the most effective
treatment ever tested at ALS TDI; the first in-human clinical trial will start enrolling in 2018.
In truth, more than one treatment will be needed to bring relief to all those diagnosed with ALS. So, while we must be all in
on AT-1501, we also must ensure that we advance other well validated compounds into trials as quickly as possible. That's why
we continue to urgently search for other exciting treatments beyond AT-1501 to advance into clinical trials. In fact, ALS TDI
screens more potential treatments in its labs each year than anywhere else in the world. We are agnostic to target, as well as
therapeutic modality. We will pursue the best ideas for slowing, stopping and ending ALS. Period.
Scientists at ALS TDI are in the process of setting up a translational research program around the next most exciting treatment
beyond AT-1501. Continuing to move the best validated ideas from the lab in to the clinic is what our mission is about; the discovery
and development of effective treatments for ALS.
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This program seeks to gain critical new insight into the discovery and clinical development of treatments for ALS through the
integration of genomic, molecular, and patient reported data.
A hallmark of chronic neurodegenerative diseases such as Alzheimer’s, Parkinsons disease, and Huntington’s is the abnormal accumulation of toxic, misfolded cellular proteins. In ALS genetic, biochemical and pathology data from animal models and patients have demonstrated that the accumulation of misfolded proteins causes neuronal stress and the activation of the unfolded protein response (UPR) is a protective mechanism.
The translation and processing of proteins is a highly regulated cellular function. Proteins that are destined for secretion or insertion into cellular membranes are translocated to a specific sub cellular organelle called the endoplasmic reticulum (ER). Under normal physiologic conditions the endoplasmic reticulum associated degradation (ERAD) system in the ER is designed to shuttle improperly folded proteins from the ER to the cytosol for degradation via the ubiquitin-proteosome system. The homeostasis of properly folding proteins in the ER, glycosylating secreted proteins, and properly inserting them into membranes prior to secretion or subcellular organelles is orchestrated by dozens of protein chaperones and enzymes. One of the must abundant and critical ER chaperones that maintains this homeostasis is the chaperone BiP/Grp78.
BiP associates with three ER transmembrane proteins ATF6, Ire1, and PERK that mediate distinct downstream UPR signaling events. Initial evidence suggested that the accumulation of unfolded proteins in the lumen of the ER disrupted a stoichiometry that exists between binding of BiP to these signaling molecules on the ER membrane versus its association with unfolded proteins. Under normal physiological conditions BiP associates with these signaling molecules keeping them tethered to the ER membrane but when the concentration of unfolded proteins in the ER increases BiP is pulled away from these molecules allowing them to be cleaved from the ER membrane and mediate downstream UPR signaling. It is now more evident that BiP does play this role to some extent but that the signaling molecules themselves are capable binding to misfolded proteins in the ER and initiating downstream UPR signaling in the absence of BiP binding.
These three regulatory proteins (ATF6, Ire1, and PERK) work together to decrease cellular stress under condition of protein misfolding by increasing the protein chaperone machinery, increasing the protein degradation machinery, and decreasing protein synthesis until cellular stress is relieved.
IRE1 is a type I ER transmembrane protein with a cytoplasmic kinase and RNAse domains. IRE1 forms oligomers under conditions of UPR activation resulting in the functional activation of its RNAse functionality. IRE1 homo oligomers dissociate from the ER plasma membrane into the cytosol. The activation of IRE1’s RNAse activity in the cytosol results in the splicing of the Xbp1 mRNA transcript into an alternatively spliced form that is recognized by the protein translational machinery which now translates Xbp1 into a functional transcription factor that enters the nucleus and changes the expression of UPR associated target genes.
PERK is a type I transmembrane protein of the ER whose cytosolic domain has a kinase domain. Under conditions of ER stress PERK dissociated from the ER membrane and its kinase activity phosphorylates the translation initiation factor EIF2A. Phosphorylation of EIF2A results in an overall reduction in protein translation in an attempt to alleviate the accumulation of misfolded proteins. A subset of RNAs are preferentially recognized by phosphortylated EIF2A however and become translated into functional proteins. These include the transcription factors CHOP and ATF4 as well as GADD34 which is a phosphatase the provides a negative feedback loop dephosphorylating EIF2A. Under prolonged conditions of ER stress the downstream targets of the transcription factor CHOP become activated and are proapoptotic genes that result in cell death.
ATF6 is a type II transmembrane protein that under conditions of ER stress translocates to the Golgi where the amino terminus is cleaved releasing an active transcription factor that enters the nucleus upregulating UPR associated genes.
In addition to its role in cleaving Xbp1 prolonged activation of the UPR results in IRE1 mediated activation of ASK1 kinase. ASK1 is a critical component of two cellular stress pathways the Jun N Terminal Kinase (JNK) Pathway and the p38 Map kinase pathway both of which activate downstream apoptotic signaling.
Several conflicting reports have suggested that either activation or inhibition of the UPR can ameliorate apoptosis and improve disease progression and improve survival in murine models of ALS. In addition several studies have demonstrated that the UPR is up regulated in post mortem spinal cords from ALS patients. Modulation of this critical pathway has shown therapeutic potential in models of prion disease, and Alzheimers disease.
At the ALS TDI we are exploring multiple ways to mediate down stream apoptotic signaling by controlling long term UPR activation. These approaches are focused on small molecule inhibitors of critical regulators of the pathway.
ALS is a progressive neuromuscular disease characterized by muscle atrophy and paralysis. One of the hallmarks of ALS that differentiates the disorder from muscular dystrophis, which primarily effects muscle fiber formation and integrity, is progressive degeneration of the synaptic connection between the motor neuron and the specific sites on muscle fibers. These highly specialized synapses are called the neuromuscular junctions (NMJs).
In ALS patients is has been demonstrated that at the time of symptom onset more than 50% of the NMJs are lost in an affected muscle group. These data have been validated in animal models of ALS where loss of NMJs occur at least thirty days prior to symptom onset.
The formation of NMJs occurs during embryogenesis and later developmental stages as the fetus actively moves in utero. It is during this period that motor units on the muscle fibers become innervated from multiple motor neurons. Postnatally a normal process of synaptic pruning occurs eliminating "weaker" connections such that by adulthood a motor unit is innervated by a single motor neuron.
In the context of injury to the muscle or nerve, the synaptic connection at the NMJ can be transiently lost. Yet the system demonstrates plasticity allowing for reinnervation by the previous motor neuron or even adjacent motor neurons. It is currently unclear how this plasticity is maintained and what signals dictate the reinnervation process.
The formation of NMJs is dependent on a complex signalling cascade that coordinates the formation of a mature synaptic cleft between muscle and the nerve terminus. A key regulator of signalling on the muscle (postsynaptic) side is the receptor tyrosine kinase MuSK. MuSK expression is pre patterned in the motor unit and is regulated by Wnt signaling and the expression of the MuSK ligand LPR4. On the nerve (presynaptic) side the expression of the LRP4 receptor Agrin by the incoming nerve terminal of the motor neuron results in the binding of Agrin to LRP4 enhancing its association with its ligand MuSK resulting in MuSK phosphorylation and clustering of MuSK-LRP4-Agrin. Phosphorylated MuSK results in the intracellular recruitment of DOK-7. In the presence of Agrin Dok-7 becomes phosphorylated resulting in further phosphorylation of MuSK and MuSK dimerization. Dimerization events result in the recruitment of adapter and anchoring proteins Rho, Rac, and rapysin all of which are required for NMJ formation and stabilization. The recruitment of the adapter proteins causes the recruitment and clustering of acetylcholine receptors. The subsequent clustering of the acetylcholine receptors results in the establishment of a highly synchronized neurotransmitter release mechanism at the NMJ synapse that converts the intracellular signaling into electrical action potential and ultimately kinetic muscle movement.
Though it is an active area of research the cellular mechanisms underlying the repair and plasticity in the context of injury and neurodegeneration such as in ALS is currently unclear. However several neurotrophic factors (IGF1, GDNF), microRNAs (mir-206), and Schwann cell derived factors (NOGO-A, Sema3A) appear to play both positive and negative roles in repair and stabilization of the NMJ. ALS TDI has several small molecule and protein therapeutic strategies targeting these key mediators of NMJ repair in the context of neurodengeration.
Recent evidence suggests that Amyotrophic Lateral Sclerosis is a non cell autonomous disease whose onset and progression are influenced by the immune system.
Microglial activation, astrocytosis and the presence of infiltrating inflammatory cells from the periphery have been well described. There is accumulation of IgG immunoreactive deposits in the spinal cord of ALS, infiltration of lymphocytes, dendritic cells, monocytes, and macrophages into the spinal cord in ALS.
Although the role of infiltrating is poorly understood recent work would suggest that type I macrophages are localized to neurons that have "died" back from their appropriate connections on muscle fibers, and pro inflammatory lymphocytes are present in the periphery both of which infiltrate into central nervous system tissues. The infiltration of pro inflammatory cell types results in astrocyte and microglial activation with ensuing neuroinflammatory cascades.
It is generally accepted that two signals are required for maximal T cell activation. The engagement of the T Cell Receptor (TCR) on T cells with antigen bound Major Histocompatibility Complex (MHC) on antigen presenting cells (APCs) and binding of CD28 on T cells to B7 receptors on APCs. With the development of knock out mice and blocking antibodies it became apparent that there were multiple CD28 homologues capable of binding B7 receptors on APCs and that multiple B7 receptors existed with different affinities for CD28 ligands/receptors. CTLA4 a homologue of CD28, is expressed on activated T cells, has 20 times higher affinity than CD28 for binding to B7 on APCs, and blocks T cell activation. A soluble form of CTLA4 (CTLA4-Ig is capable of binding B7 receptors and blocking T cell activation. Expression cloning identified two distinct B7 receptors on APSs, B7-1 (CD80) and B7-2 (CD86) both of which are capable of binding CD28 and CTLA4.
In addition to the B7 receptors the Tumor Necrosis Factor (TNF) family member CD40 is up regulated on APCs after activation by foreign antigens. In CD40 knockout mice the activation of APCs by foreign antigen up regulates CD80, CD86, and MHC expression on APCs but fails to activate T cells or elicit a humoral response. CD40 is expressed on mature B cells, macrophages, and dendritic cells. The receptor for Cd40, CD40L (CD154, gp39) is a Type I membrane receptor and a member of the TNFR superfamily. CD40L expressed on activated T cells as a result of antigen presentation. CD40 ligation induces expression of adhesion molecules and costimulatory molecules on APCs such as CD44, ICAM1, ITGAX (CD11c ), CCL3 (Mip1a); Fcgr2b (CD32); ITGAM (CD11b); ITGB2 (LFA1/CD18) and MHC class II molecules.
Induction of CD40L expression on T cells activates B cells which can be blocked by antiCD40L Abs (MR1). Administration of anti CD40L Ab MR1 blocks primary humoral responses in vivo induced by the immunization of SRBC (Sheep red blood cells) as assessed by the number of plaque forming units in the spleen of treated mice. Transient treatment with MR1 results in prolonged inhibition of primary humoral response in vivo including the expression of immunoglobulin isotypes.
A significant body of work has demonstrated the immunomodulatory effects of blocking CD40L, CD80 and CD86, or both in preclinical models of transplantation and autoimmunity. Blocking CD40L function with blocking antibodies or adenoviral expression of CD40L-Ig improves allograft transplant by 30 to 90 days. Similar studies blocking CD80/CD86 on APCs with CTLA4-Ig or adenoviral expression of CTLA4-Ig transiently improves allograft transplant survival. Transplant rejection in these models is transient and graft rejection ensues over time. Longer term repression of transplant rejection can be accomplished by blocking both the costimulatory pathway with CTLA4-Ig and blocking CD40L activation of APCs with aCD40L antibodies. However secondary induced tolerance and the acceptance of a second graft has not been successful. Blocking antibodies to CD40L or genetic deletion of CD40L in mice has demonstrated that CD40L ameliorates disease progression, survival, and surrogate markers of disease in preclinical models of experimental allergic encephalomyelitis (EAE) a model of multiple sclerosis, collagen induced arthritis, and systemic lupus erythematosus.
We have recently characterized the activation of the costimulatory pathway in the spinal cord, sciatic nerve, and skeletal muscle of SOD1G93A mice using whole genome transcriptional profiling . We further demonstrated that in the periphery the costimulatory signature is associated with the accumulation of CD68+ monocytes in sciatic nerve and focal activation in the central nervous system . The activation of the costimulatory pathways was also evident in the peripheral blood in 47% of ALS patients analyzed. In an effort to determine whether anti-CD40L would impact disease progression in the SOD1G93A mouse model, we administered an anti-CD40L monoclonal antibody, MR1, to SOD1G93A mice. Treatment with anti-CD40L delayed disease onset, improved body weight maintenance, and extended survival in this murine model of ALS. MR1 treatment reduced the number of CD68+ cells in sciatic nerve by 42%, reduced astrocyte activation in the spinal cord, improved motor neuron survival, and decreased the activation of the costimulatory pathway in spinal cord and peripheral blood.
In summary, there is considerable evidence indicating that neuroinflammation and break down of the blood brain barrier are associated with disease progression in ALS. The modulation of the costimulatory and humoral response has proven to be efficacious in preclinical models of allograft transplant, experimental allergic encephalomyelitis, collagen induced arthritis, and systems lupus erythematosis. We have previously demonstrated that modulation of the costimulatory pathway is also efficacious in ALS. Our current immune modulatory portfolio consists of several protein biologics as well as antisense oligonucleotide that should modulate the activation of the pro inflammatory cascade and provide neuroprotective benefit.
Superoxide Dismutase 1 (SOD1) is a 16kd protein which acts as an enzyme whose primary function is to scavenge and convert damaging superoxide free radicals to less damaging chemicals. SOD1 is predominantly found in the cytoplasm, but can also be detected in mitochondria and cellular nuclei. Normal SOD1 forms a 32 kd homodimer forming an 8 key beta barrel that binds a catalytic copper ion and structural zinc ion. SOD1 is an abundant and ubiquitous protein, however its cellular concentration is significantly higher in metabolic tissues and in the central nervous system. The half life of wild-type SOD1 protein is long, notably in motor neurons. The longer half-life increases the likelihood that SOD1 protein may be subjected to post translational modifications oxidation.
Mutations in the gene encoding SOD1 are associated with familial ALS. Much evidence suggests that mutant SOD1 mediated toxicity resulting in neurodegeneration is caused by some unknown gain of function by the protein. Biochemical studies have suggested that under physiological conditions even wild-type SOD1 can become oxidized and misfolded and that mutant forms of SOD1 have a higher propensity for oxidation. Further, mutant SOD1, and to a lesser extent, wild-type SOD1 has been demonstrated to oligomerize and aggregate.
It remains unclear what elements of misfolded SOD1 are deleterious. There is evidence to believe that misfolded monomeric SOD1 are damaging to cells. Alternately there is evidence that aggregated SOD1 may be toxic. The presence of both could arise from mutations or post translational modifications that promote disulfide reduction, alter the dimer interface, or adversely affect copper and zinc loading.
The shortened cellular half-life of the monomeric SOD1 when compared to the homodimer offers the basis for the hypothesis that mutant SOD1 or monomeric wild-type SOD1 are taxing to the cellular protein quality control systems resulting in less efficient cellular housekeeping by protein chaperones and protein degradation mechanisms.
There is accumulating evidence that SOD1 misfolding occurs not only in patients with mutations in the SOD1 gene but also in sporadic ALS cases. Wildtype SOD can acquire biochemical properties of mutant SOD1 via oxidative damage and several studies have detected a unique 32 kd band in post mortem spinal cord extracts from patients containing SOD1 mutations as well as sporadic ALS patients but not healthy controls or other neurodegenerative diseases.
Monoclonal antibodies and Fab fragments have also been generated that are specific for unfolded SOD1 and do not bind SOD1 dimer. These antibodies were generated using either apo SOD1 or on internal epitopes (aa 141-151) on the dimer interface of wild type SOD1 that they predicted would only be exposed on misfolded SOD1 monomers and oligomers. In SOD1 animal models these antibodies suggest that only a small percentage of total SOD1 is misfolded during disease course (less than 5%) and that misfolded SOD1 was not apparent presymptomatically. ALS TDI has worked with several of these antibodies and have shown that at least one of these antibodies does indeed slow down disease and improve survival in the SOD1G93A transgenic animal model (NI-204B, Neurimmune). ALS TDI is partnering with Neurimmune on a clinical development strategy for this antibody and investigating the ability of this antibody to detect misfolded SOD1 in sporadic ALS tissue samples.
Mutant forms of SOD1 when bound to zinc form stable dimers under physiological conditions which are stable for months and contain no oligomers. The apo (metal free) form of both WT and mutant forms of SOD1 behave very differently under normal physiological conditions across a wide temperature range. At low temperatures oligomerization occurs fairly slowly over the course of 12 months however at physiological conditions oligomerization occurs rapidly and even small increases in temperature above 15 degrees Celsius enhances this rate up to physiological temperatures. Intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The addition of reducing agents such as DTT eliminated oligomers suggesting the formation of covalent disulfide bonds during the oligomerization process. Its interesting to note that once metalated both mutant and WT SOD1 dimers are very stable and thus non toxic.
It is also interesting that in an oxidative environment the process of disulfide bond formation will be driven more aggressively under physiological conditions and that in the mitochondria which favors a more oxidative environment that the process could be greatly accelerated and favored towards oligomerization. Elevated levels of noxious monomers could arise from mutations that promote disulfide reduction, weaken the dimer interface, decrease the global stability or folding cooperativity of either the monomeric or dimeric species, or adversely affect metal loading through the copper chaperone. The low stability of the reduced monomer provides also an explanation for sporadic ALS. In these cases, impaired function of the cellular ‘‘housekeeping’’ system, i.e., chaperones, quality control, and degradation mechanisms, might cause the reduced form of wild-type SOD to enter the same cytotoxic pathway as the ALS associated mutants, consistent with observations of cytosolic SOD containing aggregates in both familial and sporadic ALS.
Proof of concept experiments have shown that a mutated multiprotein homodimer can be stabilized by the binding of a small molecule at the subunit interface. This is exemplified by that of transthyreitin. Mutations in TTR cause familial amyloid polyneuropathy (FAP). Mutations in TTR cause destabilization of the TTR homotetramer resulting in dissociation of the tetramer, monomer mis folding and protein aggregation (Connolly, 2010). The natural ligand of TTR, thyroxine, can bind to the tetramer and stabilize the tetramer. Studies identified that the destabilized tetramer creates two binding sites that when occupied by thyroxine stabilize the tetramer. A structure based design identified several small molecule scaffolds that were capable of binding to the tetramer at the T4 binding sites and stabilizing the tetramer much like the natural ligand.
Similar proof of concept experiments have shown that small molecules can stabilize the mutant SOD1 dimer driving equilibrium away from a toxic misfolded SDO1 monomer. These small molecules lack pharmacological properties for in vivo testing thus requiring medicinal chemistry for further validation in vivo. One program we are pursuing at ALS TDI is to expand on this approach and screen for small molecules that eliminate the toxicity of misfolded SOD1 via interactions with crtical epitopes on the SOD1 dimer.
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