Regulated Induced Proximity Targeting Chimeras (RIPTACs): a Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively.

While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities. Here, we describe a proof-of-concept study using novel heterobifunctional small molecules called Regulated Induced Proximity Targeting Chimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein. Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

Alzheimer risk gene product Pyk2 suppresses tau phosphorylation and phenotypic effects of tauopathy.

BACKGROUND: Genetic variation at the PTK2B locus encoding the protein Pyk2 influences Alzheimer's disease risk. Neurons express Pyk2 and the protein is required for Amyloid-ß (Aß) peptide driven deficits of synaptic function and memory in mouse models, but Pyk2 deletion has minimal effect on neuro-inflammation. Previous in vitro data suggested that Pyk2 activity might enhance GSK3ß-dependent Tau phosphorylation and be required for tauopathy. Here, we examine the influence of Pyk2 on Tau phosphorylation and associated pathology. METHODS: The effect of Pyk2 on Tau phosphorylation was examined in cultured Hek cells through protein over-expression and in iPSC-derived human neurons through pharmacological Pyk2 inhibition. PS19 mice overexpressing the P301S mutant of human Tau were employed as an in vivo model of tauopathy. Phenotypes of PS19 mice with a targeted deletion of Pyk2 expression were compared with PS19 mice with intact Pyk2 expression. Phenotypes examined included Tau phosphorylation, Tau accumulation, synapse loss, gliosis, proteomic profiling and behavior. RESULTS: Over-expression experiments from Hek293T cells indicated that Pyk2 contributed to Tau phosphorylation, while iPSC-derived human neuronal cultures with endogenous protein levels supported the opposite conclusion. In vivo, multiple phenotypes of PS19 were exacerbated by Pyk2 deletion. In Pyk2-null PS19 mice, Tau phosphorylation and accumulation increased, mouse survival decreased, spatial memory was impaired and hippocampal C1q deposition increased relative to PS19 littermate controls. Proteomic profiles of Pyk2-null mouse brain revealed that several protein kinases known to interact with Tau are regulated by Pyk2. Endogenous Pyk2 suppresses LKB1 and p38 MAPK activity, validating one potential pathway contributing to increased Tau pathology. CONCLUSIONS: The absence of Pyk2 results in greater mutant Tau-dependent phenotypes in PS19 mice, in part via increased LKB1 and MAPK activity. These data suggest that in AD, while Pyk2 activity mediates Aß-driven deficits, Pyk2 suppresses Tau-related phenotypes.

Fyn kinase inhibition reduces protein aggregation, increases synapse density and improves memory in transgenic and traumatic Tauopathy.

Accumulation of misfolded phosphorylated Tau (Tauopathy) can be triggered by mutations or by trauma, and is associated with synapse loss, gliosis, neurodegeneration and memory deficits. Fyn kinase physically associates with Tau and regulates subcellular distribution. Here, we assessed whether pharmacological Fyn inhibition alters Tauopathy. In P301S transgenic mice, chronic Fyn inhibition prevented deficits in spatial memory and passive avoidance learning. The behavioral improvement was coupled with reduced accumulation of phospho-Tau in the hippocampus, with reductions in glial activation and with recovery of presynaptic markers. We extended this analysis to a trauma model in which very mild repetitive closed head injury was paired with chronic variable stress over 2 weeks to produce persistent memory deficits and Tau accumulation. In this model, Fyn inhibition beginning 24 h after the trauma ended rescued memory performance and reduced phospho-Tau accumulation. Thus, inhibition of Fyn kinase may have therapeutic benefit in clinical Tauopathies.

In Vivo Synaptic Density Imaging with 11C-UCB-J Detects Treatment Effects of Saracatinib in a Mouse Model of Alzheimer Disease.

11C-UCB-J is a new PET tracer for synaptic density imaging. Recently, we conducted 11C-UCB-J PET on patients with mild cognitive impairment or early Alzheimer disease (AD) and found a 41% decrease in specific binding in the hippocampus compared with healthy subjects. We hypothesized that 11C-UCB-J may have potential to be a general biomarker for evaluating AD treatment effects via monitoring of synaptic density changes. In this study, we performed longitudinal 11C-UCB-J PET on AD mice to measure the treatment effects of saracatinib, which previously demonstrated synaptic changes with postmortem methods. Methods: Nine wild-type (WT) mice and 9 amyloid precursor protein and presenilin 1 double-transgenic (APPswe/PS1ΔE9 [APP/PS1]) mice underwent 3 11C-UCB-J PET measurements: at baseline, after treatment, and during drug washout. After baseline measurements, saracatinib, a Fyn kinase inhibitor currently in clinical development for AD treatment, was administered by oral gavage for 41 ± 11 d. Treatment-phase measurements were performed on the last day of treatment, and washout-phase measurements occurred more than 27 d after the end of treatment. SUVs from 30 to 60 min after injection of 11C-UCB-J were calculated and normalized by the whole-brain (WB) or brain stem (BS) average values as SUV ratio (SUVR(WB) or SUVR-1(BS)). Results: Hippocampal SUVR(WB) at baseline was significantly lower in APP/PS1 than WT mice (APP/PS1: 1.11 ± 0.04, WT: 1.15 ± 0.02, P = 0.033, unpaired t test). Using SUVR-1(BS) in the hippocampus, there was also a significant difference at baseline (APP/PS1: 0.48 ± 0.13, WT: 0.65 ± 0.10, P = 0.017, unpaired t test). After treatment with saracatinib, hippocampal SUVR(WB) in APP/PS1 mice was significantly increased (P = 0.037, paired t test). A trend-level treatment effect was seen with hippocampal SUVR-1(BS). Saracatinib treatment effects may persist, as there were no significant differences between WT and APP/PS1 mice after drug washout. Conclusion: On the basis of the 11C-UCB-J PET results, hippocampal synaptic density was lower in APP/PS1 mice than in WT mice at baseline, and this deficit was normalized by treatment with saracatinib. These results support the use of 11C-UCB-J PET to identify disease-specific synaptic deficits and to monitor treatment effects in AD.

Anti-PrPC antibody rescues cognition and synapses in transgenic alzheimer mice.

Objective: Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aßo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods: Potency of AZ59 was evaluated by binding to PrPC, blockade of Aßo interaction and interruption of Aßo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results: AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Interpretation: Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.

Systematic and standardized comparison of reported amyloid-ß receptors for sufficiency, affinity, and Alzheimer's disease relevance.

Oligomeric assemblies of amyloid-ß (Aß) peptide (Aßo) in the brains of individuals with Alzheimer's disease (AD) are toxic to neuronal synapses. More than a dozen Aß receptor candidates have been suggested to be responsible for various aspects of the molecular pathology and memory impairment in mouse models of AD. A lack of consistent experimental design among previous studies of different receptor candidates limits evaluation of the relative roles of these candidates, producing some controversy within the field. Here, using cell-based assays with several Aß species, including Aßo from AD brains obtained by autopsy, we directly compared the Aß-binding capacity of multiple receptor candidates while accounting for variation in expression and confirming cell surface expression. In a survey of 15 reported Aß receptors, only cellular prion protein (PrPC), Nogo receptor 1 (NgR1), and leukocyte immunoglobulin-like receptor subfamily B member 2 (LilrB2) exhibited direct binding to synaptotoxic assemblies of synthetic Aß. Both PrPC and NgR1 preferentially bound synaptotoxic oligomers rather than nontoxic monomers, and the method of oligomer preparation did not significantly alter our binding results. Hippocampal neurons lacking both NgR1 and LilrB2 exhibited a partial reduction of Aßo binding, but this reduction was lower than in neurons lacking PrPC under the same conditions. Finally, binding studies with soluble Aßo from human AD brains revealed a strong affinity for PrPC, weak affinity for NgR1, and no detectable affinity for LilrB2. These findings clarify the relative contributions of previously reported Aß receptors under controlled conditions and highlight the prominence of PrPC as an Aß-binding site.

Rescue of Transgenic Alzheimer's Pathophysiology by Polymeric Cellular Prion Protein Antagonists.

Cellular prion protein (PrPC) binds the scrapie conformation of PrP (PrPSc) and oligomeric ß-amyloid peptide (Aßo) to mediate transmissible spongiform encephalopathy (TSE) and Alzheimer's disease (AD), respectively. We conducted cellular and biochemical screens for compounds blocking PrPC interaction with Aßo. A polymeric degradant of an antibiotic targets Aßo binding sites on PrPC with low nanomolar affinity and prevents Aßo-induced pathophysiology. We then identified a range of negatively charged polymers with specific PrPC affinity in the low to sub-nanomolar range, from both biological (melanin) and synthetic (poly [4-styrenesulfonic acid-co-maleic acid], PSCMA) origin. Association of PSCMA with PrPC prevents Aßo/PrPC-hydrogel formation, blocks Aßo binding to neurons, and abrogates PrPSc production by ScN2a cells. We show that oral PSCMA yields effective brain concentrations and rescues APPswe/PS1ΔE9 transgenic mice from AD-related synapse loss and memory deficits. Thus, an orally active PrPC-directed polymeric agent provides a potential therapeutic approach to address neurodegeneration in AD and TSE.

Disease-modifying benefit of Fyn blockade persists after washout in mouse Alzheimer's model.

Alzheimer's disease remains without a disease-modifying therapy that improves symptoms after therapy withdrawal. Because no investigational agents have demonstrated disease-modifying effects clinically, we tested whether the Fyn inhibitor, saracatinib, provides persistent improvement in a transgenic model. Aged APPswe/PS1ΔE9 mice were treated with saracatinib or memantine for 4 weeks and spatial memory improved to control levels. After drug washout, there was sustained rescue of both memory function and synapse density by saracatinib, but a loss of benefit from memantine. These data demonstrate a disease-modifying persistent benefit for saracatinib in a preclinincal Alzheimer's model, and distinguish its action from that of memantine.

Silent Allosteric Modulation of mGluR5 Maintains Glutamate Signaling while Rescuing Alzheimer's Mouse Phenotypes.

Metabotropic glutamate receptor 5 (mGluR5) has been implicated in Alzheimer's disease (AD) pathology. We sought to understand whether mGluR5's role in AD requires glutamate signaling. We used a potent mGluR5 silent allosteric modulator (SAM, BMS-984923) to separate its well-known physiological role in glutamate signaling from a pathological role in mediating amyloid-ß oligomer (Aßo) action. Binding of the SAM to mGluR5 does not change glutamate signaling but strongly reduces mGluR5 interaction with cellular prion protein (PrPC) bound to Aßo. The SAM compound prevents Aßo-induced signal transduction in brain slices and in an AD transgenic mouse model, the APPswe/PS1ΔE9 strain. Critically, 4 weeks of SAM treatment rescues memory deficits and synaptic depletion in the APPswe/PS1ΔE9 transgenic mouse brain. Our data show that mGluR5's role in Aßo-dependent AD phenotypes is separate from its role in glutamate signaling and silent allosteric modulation of mGluR5 has promise as a disease-modifying AD intervention with a broad therapeutic window.

Binding Sites for Amyloid-ß Oligomers and Synaptic Toxicity.

In Alzheimer's disease (AD), insoluble and fibrillary amyloid-ß (Aß) peptide accumulates in plaques. However, soluble Aß oligomers are most potent in creating synaptic dysfunction and loss. Therefore, receptors for Aß oligomers are hypothesized to be the first step in a neuronal cascade leading to dementia. A number of cell-surface proteins have been described as Aß binding proteins, and one or more are likely to mediate Aß oligomer toxicity in AD. Cellular prion protein (PrPC) is a high-affinity Aß oligomer binding site, and a range of data delineates a signaling pathway leading from Aß complexation with PrPC to neuronal impairment. Further study of Aß binding proteins will define the molecular basis of this crucial step in AD pathogenesis.

High-throughput screening system for inhibitors of human Heat Shock Factor 2.

Development of novel anti-cancer drug leads that target regulators of protein homeostasis is a formidable task in modern pharmacology. Finding specific inhibitors of human Heat Shock Factor 1 (hHSF1) has proven to be a challenging task, while screening for inhibitors of human Heat Shock Factor 2 (hHSF2) has never been described. We report the development of a novel system based on an in vivo cell growth restoration assay designed to identify specific inhibitors of human HSF2 in a high-throughput format. This system utilizes a humanized yeast strain in which the master regulator of molecular chaperone genes, yeast HSF, has been replaced with hHSF2 with no detrimental effect on cell growth. This replacement preserves the general regulatory patterns of genes encoding major molecular chaperones including Hsp70 and Hsp90. The controlled overexpression of hHSF2 creates a slow-growth phenotype, which is the basis of the growth restoration assay used for high-throughput screening. The phenotype is most robust when cells are cultured at 25 °C, while incubation at temperatures greater than 30 °C leads to compensation of the phenotype. Overexpression of hHSF2 causes overexpression of molecular chaperones which is a likely cause of the slowed growth. Our assay is characterized by two unique advantages. First, screening takes place in physiologically relevant, in vivo conditions. Second, hits in our screen will be of medically relevant potency, as compounds that completely inhibit hHSF2 function will further inhibit cell growth and therefore will not be scored as hits. This caveat biases our screening system for compounds capable of restoring hHSF2 activity to a physiologically normal level without completely inhibiting this essential system.