Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

Morato, Nicolás M.; Le, MyPhuong T; Holden, Dylan T.; Cooks, R. Graham

doi:10.1177/24726303211047839

Cited by 27 publications

(44 citation statements)

References 123 publications

(185 reference statements)

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“…High‐throughput DESI‐MS analysis was carried out using the Purdue Make It system. Hardware, software, and operation of the system have been previously described [22,23,32,43] . Briefly, 50 nL samples are directly spotted from 384‐well plates onto DESI plates using a Biomek i7 fluid handling workstation (Beckman Coulter, Indianapolis, IN, USA) equipped with a 50‐nL floating slotted 384‐pin tool (V&P Scientific, San Diego, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…[19] In particular, DESI utilizes a charged solvent spray to impact on a sample, desorb the analytes present, and transfer them in secondary microdroplets to the mass spectrometer, where they ultimately yield desolvated ions. [18] Compared to other MS approaches utilized for studies of SULTs, such as those coupling liquid chromatography (LC) or direct flow-injection to electrospray ionization (ESI), [25][26][27][28][29] DESI confers unique advantages in terms of speed (< 1 s/sample vs. @ 10 s/ sample) and sample complexity, while preserving the chemical specificity and sensitivity of MS. [30] As a contactless method with high salt tolerance, [31] DESI can be used to directly analyze samples with high concentrations of buffers, non-volatile salts, or detergents, [21,22,32,33] conditions that are typical of biological assays but that are not compatible with traditional ESI-based MS analysis. Moreover, DESI-MS is a versatile technique that is potentially useful for the study of enzymatic reactions, [21,33] and also, and more extensively, for tissue imaging.…”

Section: High-throughput Label-free Desi-ms Assaymentioning

confidence: 99%

“…Hardware, software, and operation of the system have been previously described. [22,23,32,43] Briefly, 50 nL samples are directly spotted from 384-well plates onto DESI plates using a Biomek i7 fluid handling workstation (Beckman Coulter, Indianapolis, IN, USA) equipped with a 50-nL floating slotted 384-pin tool (V&P Scientific, San Diego, CA, USA). Up to 6,144 samples can be spotted on a single DESI plate, with center-to-center distances of ca.…”

Section: High-throughput Desi-msmentioning

confidence: 99%

“…We have recently extended desorption electrospray ionization (DESI) mass spectrometry (MS), a technique capable of analyzing complex samples in the ambient environment, [18–20] from organic reaction screening to model biological assays, both in a high throughput fashion (>1 sample per second) [21–23] . Here we demonstrate the first real application of the DESI‐MS technology for the study of an enzyme and its kinetic behavior.…”

Section: Introductionmentioning

confidence: 99%

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Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

et al. 2022

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The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3β-hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b-targeted drug discovery is the lack of robust assays compatible with high-throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label-free assay based on high-throughput (> 1 Hz) desorption electrospray ionization mass spectrometry (DESI-MS) for the direct quantitation of the sulfoconjugated product (CV < 10 %; < 1 ng analyte). The performance of this DESI-based assay was compared against a new fluorometric coupled-enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone > DHEA > cholesterol, for both the phosphomimetic and wild-type SULT2B1b forms. The novel DESI-MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics.

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Section: Methodsmentioning

confidence: 99%

Section: High-throughput Label-free Desi-ms Assaymentioning

confidence: 99%

Section: High-throughput Desi-msmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

et al. 2022

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“…The same can be inferred for downregulated genes and inhibition/antagonism. In addition, we are exploring the use of high-throughput robotic systems such as DESI-MS to generate large-scale interaction and activity data [82][83][84][85][86][87][88][89][90]. CANDO thus enables and illustrates the benefit of combining heterogeneous sources (gene, as well as protein expression, protein pathway databases, high-throughput binding, and activity data) to create novel types of interaction signatures/networks to produce design objectives that tackle complex indications.…”

Section: Limitations and Future Workmentioning

confidence: 99%

A Deep-Learning Proteomic-Scale Approach for Drug Design

et al. 2021

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Computational approaches have accelerated novel therapeutic discovery in recent decades. The Computational Analysis of Novel Drug Opportunities (CANDO) platform for shotgun multitarget therapeutic discovery, repurposing, and design aims to improve their efficacy and safety by employing a holistic approach that computes interaction signatures between every drug/compound and a large library of non-redundant protein structures corresponding to the human proteome fold space. These signatures are compared and analyzed to determine if a given drug/compound is efficacious and safe for a given indication/disease. In this study, we used a deep learning-based autoencoder to first reduce the dimensionality of CANDO-computed drug–proteome interaction signatures. We then employed a reduced conditional variational autoencoder to generate novel drug-like compounds when given a target encoded “objective” signature. Using this approach, we designed compounds to recreate the interaction signatures for twenty approved and experimental drugs and showed that 16/20 designed compounds were predicted to be significantly (p-value ≤ 0.05) more behaviorally similar relative to all corresponding controls, and 20/20 were predicted to be more behaviorally similar relative to a random control. We further observed that redesigns of objectives developed via rational drug design performed significantly better than those derived from natural sources (p-value ≤ 0.05), suggesting that the model learned an abstraction of rational drug design. We also show that the designed compounds are structurally diverse and synthetically feasible when compared to their respective objective drugs despite consistently high predicted behavioral similarity. Finally, we generated new designs that enhanced thirteen drugs/compounds associated with non-small cell lung cancer and anti-aging properties using their predicted proteomic interaction signatures. his study represents a significant step forward in automating holistic therapeutic design with machine learning, enabling the rapid generation of novel, effective, and safe drug leads for any indication.

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Simultaneous and Spontaneous Oxidation and Reduction in Microdroplets by the Water Radical Cation/Anion Pair

Qiu

Cooks

2022

Angewandte Chemie

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Microdroplets show unique chemistry, especially in their intrinsic redox properties, and to this we here add a case of simultaneous and spontaneous oxidation and reduction. We report the concurrent conversions of several phosphonates to phosphonic acids by reduction (RÀ P ! HÀ P) and to pentavalent phosphoric acids by oxidation. The experimental results suggest that the active reagent is the water radical cation/anion pair. The water radical cation is observed directly as the ionized water dimer while the water radical anion is only seen indirectly though the spontaneous reduction of carbon dioxide to formate. The coexistence of oxidative and reductive species in turn supports the proposal of a double-layer structure at the microdroplet surface, where the water radical cation and radical anion are separated and accumulated.

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Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

Cited by 27 publications

References 123 publications

Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

A Deep-Learning Proteomic-Scale Approach for Drug Design

Simultaneous and Spontaneous Oxidation and Reduction in Microdroplets by the Water Radical Cation/Anion Pair

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