The general amyloid interaction motif (GAIM) derived from the M13 phage tip protein g3p binds a wide variety of amyloid aggregates in a conformation-dependent manner (Krishnan 2014). Dimeric GAIM-Ig-fusions robustly bind and remodel Aβ42 amyloid aggregates and inhibit tau aggregate propagation in primary neuronal cells. In transgenic models of AD and tauopathy, GAIM-fusion treatment reduces Aβ plaque load, phospho-tau levels and improves cognition (Levenson 2016). In this study, we explored the mechanism of GAIM-mediated remodeling of amyloid aggregates by mutagenesis. Using this data, we designed a next generation Ig-fusion, NPT189, which in addition to showing improved binding potency to multiple aggregates, has reduced potential for immunogenicity after removal of potential T-cell epitopes. Data obtained from H/D exchange NMR studies, GAIM-peptide-based inhibition assays, TolA binding, and computational modeling was used for targeted mutagenesis of the GAIM scaffold. Stable, high expressing variants were then screened for binding to Aβ42 and Tau-K18 fibers using SPR and binding ELISA assays. High binding variants were further screened for Aβ42 fiber remodeling as well as tau and α-synuclein transmission inhibition activities. An independent mutagenesis study was carried out to sequentially eliminate four potential T-cell epitopes of GAIM. Analysis of binding and remodeling data collected from GAIM variants illuminated a novel mechanism for GAIM activity. Amyloid binding is mediated by residues facing the interdomain groove of N1 and N2 domains of GAIM. Binding specificity depends on the stability of both domains and the melting temperature for domain separation. Over-stabilization of N1 or N2 domains leads to reduced binding activity, while destabilization of the domains leads to increased non-specific binding. We hypothesize progressive binding and rearrangement of GAIM β-strands enables GAIM to remodel amyloid aggregates into non-fibrous and non-pathogenic aggregates. GAIM-Ig-fusions represent a novel class of therapeutics to treat protein misfolding disorders. Systemic administration of these molecules slows down the accumulation of amyloid plaque and intracellular tau tangles and provides cognitive benefits in transgenic animals.

Alzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-β (Aβ) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein ("NPT088") consisting of the active fragment of g3p and human-IgG1-Fc.Aged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically.NPT088-lowered Aβ plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice.These observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aβ and tau, the hallmark pathologies of AD.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40–100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

Protein misfolding that produces assemblies of toxic and transmissible aggregates is a central feature of the pathobiology of neurodegenerative diseases. A serendipitous discovery that direct exposure to filamentous bacteriophage M13 mediates reductions of both Aβ and tau deposits in brains of transgenic mouse models of Alzheimer's disease led to a search for the mechanism. We have isolated and characterized a fragment of the phage capsid protein responsible for the amyloid targeting activities of M13, and we show that an immunoglobulin fusion of this motif (Ig-GAIM) recapitulates the efficacy of M13 both in vitro and in transgenic Alzheimer's disease model mice following either intrahippocampal or chronic systemic administration. SPR, ELISA and dot blot based assays were used for binding analysis of fibers and oligomers. Fibers remodeling was shown by filter retention assay. Fiber assembly inhibition was monitored using ThT binding assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM to Aβ fibers using H/D exchange technique. Blocking of oligomer-Induced cytotoxicity using Ig-GAIM is shown as well. Efficacy in an aged Tg2576 AD mouse model was measured by Aβ ELISA and immunohistochemical Aβ plaque load in the hippocampus 7 days after a single bilateral intracranial injection of Ig-GAIM. Assays for amyloid fiber binding and remodeling, fiber assembly inhibition, and neuroprotection from cytotoxic oligomers together suggest this protein motif mediates these activities by potently preventing edge-to-edge beta strand aggregation. We also show that the GAIM activity mechanism is highly related to the natural role of bacteriophage infection mechanism. This targeting hypothesis is further substantiated by NMR H/D exchange spectroscopy of complexes of fibrilar Aβ and the capsid protein and by binding specificity studies on an array of misfolded and natively folded proteins. Collectively, these results indicate on a novel general mechanism of amyloid specific recognition, disruption and a potentially broad therapeutic modality. Systemic administration of GAIM Ig fusion reverses behavioral, biochemical and neuropathologic endpoints in both Aβ and Tau mouse models. The Ig-GAIM fusions represent a novel, potent, and specific therapeutic approach for reducing pathologic misfolded protein assemblies that are central players in neurodegenerative diseases.

NeuroPhage is developing NPT001 (filamentous bacteriophage M13) for the treatment of AD. NPT001 binds Aß with high affinity and disaggregates Aß plaque in a concentration-dependent manner in vitro and in vivo. In aged Tg2576 mice, amyloid plaque is significantly reduced after intra hippocampal injection of NPT001. The current study was conducted to determine if NPT001-induced clearance of Aß alters brain interstitial fluid (ISF) Aß levels. Microdialysis was used to assess ISF Aß in the brains of awake Tg2576 mice following intra hippocampal injection of NPT001. This technique measures a specific pool of unbound Aß that diffuses across a 38kDa MWCO membrane on the microdialysis probe. Aged female B6; SJL-Tg(APPSWE)2576Kha (Tg2576) mice were implanted with a unilateral 38-kDa MWCO microdialysis/injection probe into the hippocampus. Following recovery, six 90-min samples of microdialysis perfusion buffer were obtained to establish basal ISF Aß levels in each mouse. After basal sampling, 2ul of NPT001 or vehicle was injected into the hippocampus via the injection port at the tip of the microdialysis probe. ISF Aß samples were obtained across 5 days at 90-min increments following injection. Microdialysis samples were analyzed for Aßx-40 and Aßx-42 by sandwich ELISA. Following sampling, hippocampal Aß plaque load was quantified. ISF Aßx-40 levels did not increase significantly in NPT001-injected mice (n = 6) compared to vehicle-injected controls (n = 5). Interestingly, a slight lowering of ISF Aßx-40 fluctuating levels in NPT001-injected mice was observed across days. In a subset of mice tested (n = 2), Aßx-42 levels also did not increase following NPT001 injection. Importantly, NPT001 significantly reduced Aß plaque load by 52.5 ± 7.5% (mean ± SEM) in the injected hemisphere compared to the contralateral hemisphere in the same mice. In contrast, vehicle-injected mice had similar plaque load in the ipsilateral and contralateral hemispheres. All six mice in the NPT001-injected group had a decline in plaque load. Results indicate that ISF Aß levels, as assessed by in vivo microdialysis, did not change as a result of NPT001 injection despite a 50% reduction in Aß plaque load. NPT001 reduces Aß plaque load by a mechanism or pathway that does not involve solubilization and/or release of Aß monomers into brain ISF.

Misfolded protein aggregates diagnostic of AD include both extra-neuronal plaques and intracellular neurofibrillary tangles (NFTs). The significance of both amyloids for behavioral and cognitive deterioration is established in animal models and implicated in human disease progression. Plaques contain aggregated fibrils of amyloid-ß (fAß), and NFTs contain aggregated fibrils of microtubule-associated protein tau. Using biophysical and biochemical methods, we establish that filamentous bacteriophage M13 (NPT001) directly and potently dissociates a broad class of amyloids, including fAß, tau, yeast prions, and alpha-synuclein. We show that following a single administration to transgenic mouse models, NPT001 mediates Aß plaque and tau aggregate clearance, producing behavior and cognitive benefits, and indicating that NPT001 has broad amyloid clearance activity. Biochemical and biophysical assays for measuring interactions between M13 and fAß and other amyloid fibers include surface plasmon resonance (SPR) binding, thioflavin T (ThT) fluorescence, X-ray fiber diffraction, quantitative filter retardation, and differential detergent solubility. Transgenic animal models for testing Aß plaque or tau aggregate reductions after intracranial injection (IC; 2 μL intrahippocampal) or intracerebroventricular (ICV) infusion of NPT001, include PDAPP, APP751, Tg2576, and Tg4510 models. Hyperactivity and Y-maze tests assess behavior and spatial memory. Measures for fAß and tau aggregate reductions, M13-Aß co-localization, and for detecting microhemorrhage used established immunohistochemistry and neuropathology techniques. Direct, high affinity M13 binding to fAß (KD = 4nM) was shown by SPR. Consistent with its binding affinity, M13-mediated fAß dissociation ED50 is low nanomolar, shown both by loss of x-ray fAß diffraction and by reduction of amyloid ThT fluorescence. Dose- and time-dependent amyloid fiber dissociation were shown by quantifying loss of filter retention and increased detergent solubility of M13-treated fibers. Consistent with these in vitro data, we demonstrate in vivo co-localization of administered NPT001 and brain plaques, and potent NPT001-mediated reduction of Aß plaque (40%-70%) and tau (50%) without adverse effects within 7 days after a single IC administration into various transgenic mouse models. NPT001 is a novel approach for reducing Aß plaque and tau aggregate loads, a potential treatment strategy for Alzheimer's disease. The broad activity of NPT001 for dissociation of amyloid fibers suggests other neurodegenerative disease targets.

Diagnostic hallmarks of AD include both extracellular ß-amyloid plaques and intraneuronal neurofibrillary tangles (NFTs), accumulating over decades before symptomatic disease onset. The significance of both amyloids for neuronal toxicity and cognitive deterioration has been established in animal models and implicated in human disease progression. Intensive AD drug discovery efforts have been directed toward lowering both types of deposits. Plaques are composed of fibrils of aggregated amyloid-ß (fAß), and NFTs are composed of fibrils of aggregated microtubule-binding protein tau. Here, we demonstrate that NPT001 directly and potently dissociates fAß and other amyloid fibers. We show that following a single administration to aged, transgenic AD model mice, NPT001 mediates both Aß plaque and tau aggregate clearance, producing behavior and cognitive benefits, and indicating that NPT001 has broad amyloid clearance activity with a novel mechanism. Biochemical assays for measuring M13-fAß interactions include surface plasmon resonance (SPR) binding, thioflavin T (ThT) fluorescence, X-ray fiber diffraction, amyloid fiber filter retention, and detergent solubility assays. Transgenic animal models for testing Aß plaque load or tau aggregate reductions after intracranial injection or intracerebroventricular infusion of NPT001 include PDAPP, APP751, Tg2576, and 3xTg models. Hyperactivity and Y-maze tests assessed behavior and spatial memory. Measures for plaque load and tau aggregate reductions and M13-Aß plaque co-localization used established immunohistochemistry techniques. Direct, high affinity M13 binding to fAß (KD = 4nM) was shown by SPR. M13-induced disruption of fAß was demonstrated using x-ray fiber diffraction and ThT fluorescence assays. Dose- and time-dependent Aß dissociation were shown by quantifying the reduction of fAß filter retention and increased detergent solubility. We demonstrate in vivo co-localization of administered M13 and fAß and potent NPT001 reduction of Aß plaque (40%-70% reduction) without adverse effects within 7 days after a single IC administration into 4 aged hAPP tg AD mouse models. Similarly, NPT001 mediates significant clearance of tau aggregates from aged 3xTg mice. NPT001 is a novel agent, uniquely mediating clearance of both Aß and tau aggregates, a potential treatment strategy for Alzheimer's disease.