Novel hydrogels: are they poised to transform 3D cell-based assay systems in early drug discovery?

INTRODUCTION: Success in drug discovery remains unpredictable. However, more predictive and relevant disease models are becoming pivotal to demonstrating the clinical benefits of new drugs earlier in the lengthy drug discovery process. Novel hydrogel scaffolds are being developed to transform the relevance of such 3D cell-based in vitro assay systems. AREAS COVERED: Most traditional hydrogels are still of unknown composition and suffer significant batch-to-batch variations, which lead to technical constraints. This article looks at how a new generation of novel synthetic hydrogels that are based on self-assembling peptides are poised to transform 3D cell-based assay systems by improving their relevance, reproducibility and scalability. EXPERT OPINION: The emerging advantages of using these novel hydrogels for human 3D screening assays should enable the discovery of more cost-effective drugs, leading to improved patient benefits. Such a disruptive change could also reduce the considerable time lag from obtaining in vitro assay data to initiating clinical trials. There is now a sufficient body of data available in the literature to enable this ambition to become a reality by significantly improving the predictive validity of 3D cell-based assays in early drug discovery. Novel hydrogels are key to unlocking the full potential of these assay systems.

Predictive validity in drug discovery: what it is, why it matters and how to improve it.

Successful drug discovery is like finding oases of safety and efficacy in chemical and biological deserts. Screens in disease models, and other decision tools used in drug research and development (R&D), point towards oases when they score therapeutic candidates in a way that correlates with clinical utility in humans. Otherwise, they probably lead in the wrong direction. This line of thought can be quantified by using decision theory, in which 'predictive validity' is the correlation coefficient between the output of a decision tool and clinical utility across therapeutic candidates. Analyses based on this approach reveal that the detectability of good candidates is extremely sensitive to predictive validity, because the deserts are big and oases small. Both history and decision theory suggest that predictive validity is under-managed in drug R&D, not least because it is so hard to measure before projects succeed or fail later in the process. This article explains the influence of predictive validity on R&D productivity and discusses methods to evaluate and improve it, with the aim of supporting the application of more effective decision tools and catalysing investment in their creation.

Converging global crises are forcing the rapid adoption of disruptive changes in drug discovery.

Spiralling research costs combined with urgent pressures from the Coronavirus 2019 (COVID-19) pandemic and the consequences of climate disruption are forcing changes in drug discovery. Increasing the predictive power of in vitro human assays and using them earlier in discovery would refocus resources on more successful research strategies and reduce animal studies. Increasing laboratory automation enables effective social distancing for researchers, while allowing integrated data capture from remote laboratory networks. Such disruptive changes would not only enable more cost-effective drug discovery, but could also reduce the overall carbon footprint of discovering new drugs.

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

Novel 5-aryloxypyrimidine SEN1576 as a candidate for the treatment of Alzheimer's disease.

Prefibrillar assembly of amyloid-ß (Aß) is a major event underlying the development of neuropathology and dementia in Alzheimer's disease (AD). This study determined the neuroprotective properties of an orally bioavailable Aß synaptotoxicity inhibitor, SEN1576. Binding of SEN1576 to monomeric Aß 1-42 was measured using surface plasmon resonance. Thioflavin-T and MTT assays determined the ability of SEN1576 to block Aß 1-42-induced aggregation and reduction in cell viability, respectively. In vivo long-term potentiation (LTP) determined effects on synaptic toxicity induced by intracerebroventricular (i.c.v.) injection of cell-derived Aß oligomers. An operant behavioural schedule measured effects of oral administration following i.c.v. injection of Aß oligomers in normal rats. SEN1576 bound to monomeric Aß 1-42, protected neuronal cells exposed to Aß 1-42, reduced deficits in in vivo LTP and behaviour. SEN1576 exhibits the necessary features of a drug candidate for further development as a disease modifying treatment for the early stages of AD-like dementia.

Dose-dependent improvements in learning and memory deficits in APPPS1-21 transgenic mice treated with the orally active Aß toxicity inhibitor SEN1500.

In the Alzheimer's disease (AD) brain, accumulation of Aß1-42 peptides is suggested to initiate a cascade of pathological events. To date, no treatments are available that can reverse or delay AD-related symptoms in patients. In the current study, we introduce a new Aß toxicity inhibitor, SEN1500, which in addition to its block effect on Aß1-42 toxicity in synaptophysin assays, can be administered orally and cross the blood-brain barrier without adverse effects in mice. In a different set of animals, APPPS1-21 mice were fed with three different doses of SEN1500 (1 mg/kg, 5 mg/kg and 20 mg/kg) for a period of 5 months. Cognition was assessed in a variety of behavioral tests (Morris water maze, social recognition, conditioned taste aversion and passive avoidance). Results suggest a positive effect on cognition with 20 mg/kg SEN1500 compared to control APPPS1-21 mice. However, no changes in soluble or insoluble Aß1-40 and Aß1-42 were detected in the brains of SEN1500-fed mice. SEN1500 also attenuated the effect of Aß1-42 on synaptophysin levels in mouse cortical neurons, which indicated that the compound blocked the synaptic toxicity of Aß1-42. In vitro and in vivo effects presented here suggest that SEN1500 could be an interesting AD therapeutic.

Orally bioavailable small molecule drug protects memory in Alzheimer's disease models.

Oligomers of beta-amyloid (Aß) are implicated in the early memory impairment seen in Alzheimer's disease before to the onset of discernable neurodegeneration. Here, the capacity of a novel orally bioavailable, central nervous system-penetrating small molecule 5-aryloxypyrimidine, SEN1500, to prevent cell-derived (7PA2 [conditioned medium] CM) Aß-induced deficits in synaptic plasticity and learned behavior was assessed. Biochemically, SEN1500 bound to Aß monomer and oligomers, produced a reduction in thioflavin-T fluorescence, and protected a neuronal cell line and primary cortical neurons exposed to synthetic soluble oligomeric Aß(1-42). Electrophysiologically, SEN1500 alleviated the in vitro depression of long-term potentiation induced by both synthetic Aß(1-42) and 7PA2 CM, and alleviated the in vivo depression of long-term potentiation induced by 7PA2 CM, after systemic administration. Behaviorally, oral administration of SEN1500 significantly reduced memory-related deficits in operant responding induced after intracerebroventricular injection of 7PA2 CM. SEN1500 reduced cytotoxicity, acute synaptotoxicity, and behavioral deterioration after in vitro and in vivo exposure to synthetic Aß and 7PA2 CM, and shows promise for development as a clinically viable disease-modifying Alzheimer's disease treatment.

The N-methylated peptide SEN304 powerfully inhibits Aß(1-42) toxicity by perturbing oligomer formation.

Oligomeric forms of ß-amyloid (Aß) have potent neurotoxic activity and are the primary cause of neuronal injury and cell death in Alzheimer's disease (AD). Compounds that perturb oligomer formation or structure may therefore be therapeutic for AD. We previously reported that d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2) (SEN304) is able to inhibit Aß aggregation and toxicity, shown primarily by thioflavin T fluorescence and MTT (Kokkoni, N. et al. (2006) N-Methylated peptide inhibitors of ß-amyloid aggregation and toxicity. Optimisation of inhibitor structure. Biochemistry 45, 9906-9918). Here we extensively characterize how SEN304 affects Aß(1-42) aggregation and toxicity, using biophysical assays (thioflavin T, circular dichroism, SDS-PAGE, size exclusion chromatography, surface plasmon resonance, traveling wave ion mobility mass spectrometry, electron microscopy, ELISA), toxicity assays in cell culture (MTT and lactate dehydrogenase in human SH-SHY5Y cells, mouse neuronal cell death and synaptophysin) and long-term potentiation in a rat hippocampal brain slice. These data, with dose response curves, show that SEN304 is a powerful inhibitor of Aß(1-42) toxicity, particularly effective at preventing Aß inhibition of long-term potentiation. It can bind directly to Aß(1-42), delay ß-sheet formation and promote aggregation of toxic oligomers into a nontoxic form, with a different morphology that cannot bind thioflavin T. SEN304 appears to work by inducing aggregation, and hence removal, of Aß oligomers. It is therefore a promising lead compound for Alzheimer's disease.

Novel anti-inflammatory compound SEN1176 alleviates behavioral deficits induced following bilateral intrahippocampal injection of aggregated amyloid-ß1₋42.

Behavioral effects of a novel anti-inflammatory SEN1176 were investigated. This pyrrolo[3,2-e][1,2,4]triazolo[1,5-a]pyrimidine suppresses amyloid-ß (Aß)1-42-induced macrophage production of nitric oxide, TNF-α, IL-1ß, and IL-6 in a dose-dependent fashion, an activity profile consistent with SEN1176 being a neuroinflammation inhibitor. Using male Sprague-Dawley rats, SEN1176 was examined relative to detrimental behavioral effects induced following bilateral intrahippocampal (IH) injections of aggregated Aß1-42. The rats were trained to respond under an alternating-lever cyclic-ratio (ALCR) schedule of food reinforcement, enabling measurement of parameters of operant performance that reflect aspects of learning and memory. Under the ALCR schedule, orally administered SEN1176 at 5, 20, or 30 mg/kg was effective in reducing the behavioral deficit caused by bilateral IH aggregated Aß1-42 injections in a dose-related manner over a 90-day treatment period. SEN1176 at 20 and 30 mg/kg significantly reduced lever switching errors and, at doses of 5, 10, and 30 mg/kg, significantly reduced incorrect lever perseverations, indicating a reduction of the behavioral deficit induced as a result of inflammation following IH Aß1-42 injections. When treatment with SEN1176 was instigated 30 days after IH Aß1-42 injections, it resulted in progressive protection, and withdrawal of SEN1176 treatment 60 days after IH Aß1-42 injections revealed partial retention of the protective effect. SEN1176 also significantly reduced numbers of activated astrocytes adjacent to the aggregated Aß1-42 injection sites. These results indicate the potential of SEN1176 for alleviating chronic neuroinflammatory processes related to brain Aß deposition that affect learning and memory in Alzheimer's disease.

RS-0406 arrests amyloid-beta oligomer-induced behavioural deterioration in vivo.

Clinically accessible compounds that arrest or reverse the effects of amyloid-beta (Abeta) on progressively developing behavioural symptomatology and neuropathology in Alzheimer's disease (AD) have yet to become available. However, a viable strategy may be to target and neutralise soluble Abeta oligomers, which have been shown to mediate synaptic dysfunction and to produce cognitive deficits in the intact organism. Inhibiting the aggregation of Abeta is therapeutically attractive, as Abeta aggregation is a pathological event and pharmacological interventions targeting this are likely to have a non-toxic profile. A behavioural assay, the alternating-lever cyclic-ratio schedule, was used to assess the effect of Abeta oligomers and the non-peptide small molecule RS-0406 in male Sprague-Dawley rats. RS-0406 has been shown to inhibit Abeta(1-42) fibrillogenesis and protect against Abeta(1-42)-induced cytotoxicity in primary hippocampal neurons. In the current study, RS-0406 ameliorated the adverse effects of secreted oligomers of human Abeta on behaviour and dose dependently reduced the behavioural effects of Abeta oligomers, with the highest dose, 10microM, maintaining behaviour approximately at control levels. This effect appeared to be central; peripheral confounds having been extensively investigated. This is the first published report on the effects of RS-0406 in vivo and indicates that RS-0406 has potential as a pharmacotherapeutic intervention for behavioural deficits seen in the early stages of AD, and possibly as an intervention in the development of AD neuropathology. Indeed, an analogue of RS-0406 that could be administered peripherally might be a realistic candidate for the clinical treatment of AD.

Potassium channels: gene family, therapeutic relevance, high-throughput screening technologies and drug discovery.

Existing drugs that modulate ion channels represent a key class of pharmaceutical agents across many therapeutic areas and there is considerable further potential for potassium channel drug discovery. Potassium channels represent the largest and most diverse sub-group of ion channels and they play a central role in regulating the membrane potential of cells. Recent advances in genomics have greatly added to the number of these potential drug targets, but selecting a suitable potassium channel for drug discovery research is a key step. In particular, the potential therapeutic relevance of a potassium channel should be taken into account when selecting a target for screening. Potassium channel drug discovery is being driven by a need to identify lead compounds that can provide tractable starting points for medicinal chemistry. Furthermore, advances in laboratory automation have brought significant opportunities to increase screening throughput for potassium channel assays, but careful assay configuration to model drug-target interactions in a physiological manner is an essential consideration. Several potassium channel screening platforms are described in this review in order to provide some insight into the variety of formats available for screening, together with some of their inherent advantages and limitations. Particular emphasis is placed on the mechanistic basis of drug-target interaction and those aspects of structure/function that are of prime importance in potassium channel drug discovery.