The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via β-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the β strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM-hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aβ plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates.

The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via β-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the β strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM–hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aβ plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates. © 2019 Elsevier Ltd. All rights reserved.

Therapeutic strategies that target pathways of protein misfolding and the toxicity of intermediates along these pathways are mainly at discovery and early development stages, with the exception of monoclonal antibodies that have mainly failed to produce convincing clinical benefits in late stage trials. The clinical failures represent potentially critical lessons for future neurodegenerative disease drug development. More effective drugs may be achieved by pursuing the following two strategies. First, conformational targeting of aggregates of misfolded proteins, rather than less specific binding that includes monomer subunits, which vastly outnumber the toxic targets. Second, since neurodegenerative diseases frequently include more than one potential protein pathology, generic targeting of aggregates by shape might also be a crucial feature of a drug candidate. Incorporating both of these critical features into a viable drug candidate along with high affinity binding has not been achieved with small molecule approaches or with antibody fragments. Monoclonal antibodies developed so far are not broadly acting through conformational recognition. Using GAIM (General Amyloid Interaction Motif) represents a novel approach that incorporates high affinity conformational recognition for multiple protein assemblies, as well as recognition of an array of assemblies along the misfolding pathway between oligomers and fibers. A GAIM-Ig fusion, NPT088, is nearing clinical testing.

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.

The accumulation of combinations of aggregated amyloid-β, tau, and α-synuclein within a single disease, such as found in Alzheimer's disease (AD), evokes therapeutic strategies that generically target aggregates, independent of primary protein sequence. NPT088 is a human immunoglobulin (huIgG1Fc) fusion that displays two copies of the General Amyloid Interaction Motif (GAIM). We have shown that NPT088 has uniquely broad activities, both in vitro and in vivo, against multiple neuropathological aggregates, making it a novel candidate for treating AD. NPT088 measured for amyloid (Aβ, tau, α-synuclein) binding and binding specificity using ELISA and SPR formats. Aggregation inhibition monitored by ThT. Oligomer-induced cytotoxicity, measured on N2a cells using adenylate kinase release assay. Aβ aggregate-NPT088 co-precipitation assays used aged Tg2576 brain homogenates. NPT088 administered to Tg AD and PD model mice weekly i.p., 14 weeks: Tg2576,18 m.o.; rTg4510, 3.6 m.o.; mThy1-Hα-synuclein,3.2 m.o.. Cognition assessed by spontaneous alternation (Y-maze) and novel object recognition. Brain biochemistry performed using Western or ELISA on soluble and insoluble fractions from homogenates or CSF. Neuropathology utilized 40μm fixed sections. NPT088 specifically and potently binds amyloid fibers of Aβ, tau and α-synuclein (Kds =5-46 nM), but does not bind monomers or natively aggregated proteins. NPT088 binds Aβ oligomers, blocks oligomer-induced cytotoxicity (IC50<5nM), and prevents Aβ and tau aggregation. NPT088 recognizes brain homogenate Aβ aggregates from aged Tg2576 mice. NPT088 effectively treats Tg AD and PD mouse models. In the Tg2576 hAPP model, NPT088 significantly: improves cognition; reduces brain Aβ(1-42) and Aβ plaque; and reduces Aβ in CSF. In rTg4510 tau mice, NPT088 significantly: improves cognition, reduces levels of phospho-tau associated with neuropathology and lowers CSF tau. In mThy1-Hα-synuclein mice, NPT088 significantly reduces proteinase-KRα-synuclein and increases tyrosine hydroxylase levels. NPT088 has successfully completed a 1-month exploratory monkey safety study. These results demonstrate that NPT088 is a first-in-class therapeutic candidate for AD, which targets misfolded proteins generically, including aggregates of both Aβ and tau. Following IND filing in 4Q2015, NPT088 will be tested for proof of activity in AD by measuring reduction of PET amyloid markers.

Alzheimer's disease (AD) is characterized by both Amyloid b (A b) plaque deposition and intracellular neurofibrillary tangles accompanied by progressive cognitive decline. We isolated a protein motif from M13 bacteriophage that binds to and disaggregates multiple amyloids, which we call the General Amyloid Interaction Motif or GAIM. The present studies were conducted to evaluate whether NPT088, a Hu-IgG 1 -Fc fusion protein bivalent for GAIM was able to mediate the clearance of A b plaques and reduction of phospho-Tau (pTau) in transgenic AD mouse models when administered systemically. NPT088 or PBS was administered weekly via intraperitoneal injection for 10-14 weeks. Mice were aged Tg2576 (21-25 mo) and rTg4510 mice (6.5-7 mo) at the end of dosing. Behavior (activity and cognition) was assessed at various times throughout experiments. In Tg2576 mice, Aβ was measured by ELISA for Aβ 42 in RIPA (soluble) and formic acid (insoluble) fractions of frontal cortex and hippocampus; neuropathology was quantified using both 82E1 immunohistochemistry and thioflavin S histochemistry. In rTg4510 mice, pTau (AT8 & pSer422) was quantified biochemically by western blot from several fractions of brain; AT8 immunoreactivity was quantified in hippocampus. Chronic, systemic administration of NPT088 to Tg2576 mice significantly reduced hyperactivity, increased spontaneous alternation and improved performance in novel object recognition within 3 months of treatment. Levels of A b 42 were significantly reduced in cortex and hippocampus as measured by ELISA. Immunohistochemistry indicated a significant reduction of A b plaques in hippocampus, and these reductions were not associated with microhemorrhage. Chronic systemic administration of NPT088 to rTg4510 mice significantly improved performance in novel object recognition. Levels pTau (AT8, pSer422) were significantly reduced in a high-salt, low-speed insoluble fraction from cortex that is enriched for neurofibrillary tangles. pSer422 and AT270 pTau were also reduced in other, more soluble fractions. Immunohistochemistry indicated significant reduction of pTau (AT8) in the hippocampus. Collectively, these results indicate that chronic (3 month) systemic administration of NPT088 reverses behavioral, biochemical and anatomical pathologies in both A b and Tau mouse models. These data support the use of NPT088 as a novel therapeutic targeting multiple misfolded proteins for the treatment 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.

Alzheimer's disease (AD) is characterized by both Amyloid b (Aβ) plaque deposition and intracellular neurofibrillary tangles accompanied by progressive cognitive decline. We have shown that NPT002 (filamentous bacteriophage M13) dose-dependently clears Aβ plaques and restores normal cognitive performance in aged Tg2576 mice. Recently, we isolated the protein motif from NPT002 responsible for the amyloid-interacting activity, which we call the generic amyloid interacting motif or GAIM. The present study was conducted to evaluate whether fusion proteins bivalent for GAIM were able to clear β-amyloid aggregates in transgenic Alzheimer's mouse models. In the present study, we administered test article or control treatment via a single intra-hippocampal 2μL injection per hemisphere (7–8μg of protein per injection). Mice used for these studies included 17–19 month old male Tg2576 mice and 17–19 month old male & female 3xTg mice. Animals were sacrificed 7 days post-treatment. Paraformaldehyde-fixed brains were serially sectioned and analyzed for Aβ load in the hippocampus and surrounding cortical areas by ThioS fluorescence and anti-amyloid antibody staining (82E1). Neuro-inflammation was monitored by antibody staining for Iba1 and GFAP. Synaptic density was assessed by antibody staining for synaptophysin (SY38). Systemic administration of NPT088 was via intraperitoneal injection at 10mg/kg. Intrahippocampal injection of either NPT014 (human IgG 4 -Fc-GAIM) or NPT088 (human IgG 1 -Fc-GAIM) into aged Tg2576 mice resulted in significantly lower β-amyloid plaque in hippocampus and surrounding neocortex 7 days after treatment as measured by either 82E1 or ThioS. The decrease in β-amyloid was accompanied by significant increases in Iba-1 and synaptophysin. GFAP and Perl's Prussian Blue staining was unaffected by treatment. Similar to observations in aged Tg2576 mice, intra-hippocampal injection of NPT088 into aged 3xTg mice resulted in significant decreases in β-amyloid in the hippocampus. The timing and magnitude of the effects of NPT088 and NPT014 on β-amyloid were similar to previous studies with NPT002. Moreover, acute systemic administration of NPT088 to wt mice produced brain penetration. NPT088 and NPT014 safely reduced β-amyloid plaque and increased synaptic density within 7 days of direct-to-brain administration in transgenic mouse models. These data support the use of GAIM-containing molecules as a novel therapeutic approach for reducing Aβ plaque.

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.

We attempted to engineer a novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude supraphysiological concentrations shortly after injection; (ii) maintenance of low-circulating level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating insulin through its amino side chains to a 40kDa polyethylene glycol containing sulfhydryl moiety (PEG(40)-SH), employing recently developed hetero-bifunctional spacer 9-hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc-0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by spontaneous chemical hydrolysis has been created. PEG(40)-Fmoc-insulin is a water-soluble, reactivatable prodrug with low biological activity. Upon incubation at physiological conditions, the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear fashion, releasing the nonmodified immunologically and biologically active insulin with a t(1/2) value of 30h. A single subcutaneous administration of PEG(40)-Fmoc-insulin to healthy and diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar doses of the native hormone. The beneficial pharmacological features endowed by PEGylation are thus preserved. In contrast, nonreversible, "conventional" pegylation of insulin led to inactivation of the hormone.

This paper reports the use of mass spectrometry to characterize oligonucleotides immobilized to the surfaces of biochips. Biotinylated oligonucleotides were immobilized to self-assembled monolayers that present a streptavidin layer and then treated with a complementary strand to present short duplexes. Treatment of the surface with 5-methoxysalicylic acid and ammonium citrate matrix allows the individual oligonucleotides to be observed by matrix-assisted laser desorption/iozation and time-of-flight mass spectrometry (MALDI-TOF MS). Examples are shown wherein this method is applied to assays of hybridization, of cleavage by a deoxyribozyme, of a dephosphorylation reaction, and of the adducts formed on treatment of DNA with cis-platin. This work provides an early example of the application of mass spectrometry to DNA biochips and may substantially expand the applications of the now common oligonucleotide arrays.