Technologies that enable rapid screening of diverse reaction conditions are of critical importance to methodology development and reaction optimization, especially when molecules of high complexity and scarcity are involved. The lackofageneral solid dispensing method for chemical reagents on micro-and nanomole scale prevents the full utilization of reaction screening technologies.W eherein report the development of at echnology in whichg lass beads coated with solid chemical reagents (ChemBeads) enable the delivery of nanomole quantities of solid chemical reagents efficiently.B y exploring the concept of preferred screening sets,the flexibility and generality of this technology for high-throughput reaction screening was validated.High-throughput reaction screening is at ool that enables the investigation of large numbers of reaction conditions in parallel for ap articular chemical transformation. Reaction screening is an essential component for any new synthetic methodology development and is often utilized in complex natural product total synthesis.Whenconducted in parallel, it has the potential to offer high speed and efficiency in identifying an ovel or optimal reaction condition. Importantly,itisideal to miniaturize the reaction scale so that only milligram quantities of high-value intermediates are consumed when running arrays of reaction conditions. [1] Historically,the advancement and broad impact of high-throughput reaction screening has been hindered by al ack of suitable technologies to effectively handle reaction miniaturization. Only in the last few years have transformative advancements in this field started to emerge.S eminal papers from Merck and Pfizer have demonstrated the use of bioassay equipment and flow instrumentation to enable nanomole scale reaction screening in ah igh-throughput fashion. [2] However, ac ritical field-wide challenge that has yet to be addressed is how to effectively dispense diverse solid chemical reagents on nanomole (submilligram) scale accurately and efficiently. [3] Our search for ar obotic platform capable of dispensing av ariety of solids reagents in small quantities (< 1mg) was unsuccessful. [4] Each reagent would require individualized protocol development for accurate robotic dispensing, making it unrealistic to have as ingle platform for all solids. [2b,3b] As ar esult, tedious manual weighing has been the only reliable method. With nanomole quantities of material, this becomes unfeasible.T hus,n anomole scale reaction screening relies almost exclusively on the use of stock solutions made from reagents which are soluble in the reaction solvent. [2,5] Ideally, any combination of reagents,i nitially soluble or not, need to be incorporated in as creening set in order to have an unbiased assessment of reactivity.I nl ight of this field-wide challenge,w ee ndeavored to develop au niversal method for dispensing solid chemical reagents on nanomole scale with accuracy and efficiencyi nt he context of high throughput reaction screening.During our research, we became awa...
High-throughput experimentation is a technique for screening multiple reaction conditions in parallel at micro or nanoscale without depleting precious starting materials. However, assembling a comprehensive screening set often involves the distribution of large number of solid reagents with diverse physical properties in small quantities. Automated solid dispensing, especially at submilligram scale, has long been a challenge with no practical and reliable solutions. This paper describes the use of our newly developed chemical-coated beads technology to provide a universal approach to the solid handling problem. This technology, when combined with an automated solid dispensing platform or calibrated scoops, can dispense submilligram quantities of a variety of solids with efficiency and adequate accuracy.
Background Upper motor neurons (UMNs) are a key component of motor neuron circuitry. Their degeneration is a hallmark for diseases, such as hereditary spastic paraplegia (HSP), primary lateral sclerosis (PLS), and amyotrophic lateral sclerosis (ALS). Currently there are no preclinical assays investigating cellular responses of UMNs to compound treatment, even for diseases of the UMNs. The basis of UMN vulnerability is not fully understood, and no compound has yet been identified to improve the health of diseased UMNs: two major roadblocks for building effective treatment strategies. Methods Novel UMN reporter models, in which UMNs that are diseased because of misfolded superoxide dismutase protein (mSOD1) toxicity and TDP‐43 pathology are labeled with eGFP expression, allow direct assessment of UMN response to compound treatment. Electron microscopy reveals very precise aspects of endoplasmic reticulum (ER) and mitochondrial damage. Administration of NU‐9, a compound initially identified based on its ability to reduce mSOD1 toxicity, has profound impact on improving the health and stability of UMNs, as identified by detailed cellular and ultrastructural analyses. Results Problems with mitochondria and ER are conserved in diseased UMNs among different species. NU‐9 has drug‐like pharmacokinetic properties. It lacks toxicity and crosses the blood brain barrier. NU‐9 improves the structural integrity of mitochondria and ER, reduces levels of mSOD1, stabilizes degenerating UMN apical dendrites, improves motor behavior measured by the hanging wire test, and eliminates ongoing degeneration of UMNs that become diseased both because of mSOD1 toxicity and TDP‐43 pathology, two distinct and important overarching causes of motor neuron degeneration. Conclusions Mechanism‐focused and cell‐based drug discovery approaches not only addressed key cellular defects responsible for UMN loss, but also identified NU‐9, the first compound to improve the health of diseased UMNs, neurons that degenerate in ALS, HSP, PLS, and ALS/FTLD patients.
Technologies that enable rapid screening of diverse reaction conditions are of critical importance to methodology development and reaction optimization, especially when molecules of high complexity and scarcity are involved. The lack of a general solid dispensing method for chemical reagents on micro‐ and nanomole scale prevents the full utilization of reaction screening technologies. We herein report the development of a technology in which glass beads coated with solid chemical reagents (ChemBeads) enable the delivery of nanomole quantities of solid chemical reagents efficiently. By exploring the concept of preferred screening sets, the flexibility and generality of this technology for high‐throughput reaction screening was validated.
Even though amyotrophic lateral sclerosis (ALS) is a disease of the upper and lower motor neurons, to date none of the compounds in clinical trials have been tested for improving the health of diseased upper motor neurons (UMNs). There is an urgent need to develop preclinical assays that include UMN health as a readout. Since ALS is a complex disease, combinatorial treatment strategies will be required to address the mechanisms perturbed in patients. Here, we describe a novel in vitro platform that takes advantage of an UMN reporter line in which UMNs are genetically labeled with fluorescence and have misfolded SOD1 toxicity. We report that NU-9, an analog of the cyclohexane-1,3-dione family of compounds, improves the health of UMNs with misfolded SOD1 toxicity more effectively than riluzole or edaravone, -the only two FDA-approved ALS drugs to date-. Interestingly, when NU-9 is applied in combination with riluzole or edaravone, there is an additive effect on UMN health, as they extend longer axons and display enhanced branching and arborization, two important characteristics of healthy UMNs in vitro.
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