High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants

Aditham, Arjun K.; Markin, Craig J.; Mokhtari, Daniel A.; DelRosso, Nicole; Fordyce, Polly M.

doi:10.1016/j.cels.2020.11.012

Cited by 27 publications

(44 citation statements)

References 108 publications

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“…A full description of materials and methods is provided in the supplementary materials. Briefly, we fabricated microfluidic devices and aligned them to plasmid DNA arrays as previously described (70)(71)(72), then connected devices to a custom pneumatic manifold (73) and imaged using a fully automated fluorescence microscope. The plasmid DNAs coded for a set of specified PafA variants, each with a C-terminal eGFP tag.…”

Section: Materials and Methods Summarymentioning

confidence: 99%

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Markin

Mokhtari

Sunden

et al. 2021

Science

132

136

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Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

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Section: Materials and Methods Summarymentioning

confidence: 99%

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Markin

Mokhtari

Sunden

et al. 2021

Science

132

136

View full text Add to dashboard Cite

show abstract

“… 49 Future approaches to further explore degradation opportunities may require an in-depth kinetic analysis of protein complexes as those used for dynamic transcription factors, 37 which microfluidics and single molecule studies may also be well positioned to address. 50 Drawing from enzymology principles, a deeper appreciation and mechanistic understanding of energetics for protein ensembles could provide new avenues for protein degradation paradigms. 51 − 54 Productive integration of these molecular contributions will continue to refine kinetic degradation models and translational frameworks to successfully develop bivalent degraders.…”

Section: Induced Cooperativity and Pursuit Of Privileged Ternary Complexes For Proteasomal Degradationmentioning

confidence: 99%

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

Rodriguez‐Rivera

Levi

2021

ACS Cent. Sci.

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Diverging from traditional target inhibition, proteasomal protein degradation approaches have emerged as novel therapeutic modalities that embody distinct pharmacological profiles and can access previously undrugged targets. Small molecule degraders have the potential to catalytically destroy target proteins at substoichiometric concentrations, thus lowering administered doses and extending pharmacological effects. With this mechanistic premise, research efforts have advanced the development of small molecule degraders that benefit from stable and increased affinity ternary complexes. However, a holistic framework that evaluates different degradation modes from a catalytic perspective, including focusing on kinetically favored degradation mechanisms, is lacking. In this Outlook, we introduce the concept of an induced cooperativity spectrum as a unifying framework to mechanistically understand catalytic degradation profiles. This framework is bolstered by key examples of published molecular degraders extending from molecular glues to bivalent degraders. Critically, we discuss remaining challenges and future opportunities in drug discovery to rationally design and phenotypically screen for efficient degraders.

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“…Two recently introduced platforms-STAMMP (Aditham et al, 2021) and HT-MEK (Markin et al, 2020) -leverage microfluidic devices containing >1,500 valved reaction chambers to allow for parallel on-chip expression and purification of fluorescently labeled proteins. After expression, proteins can be recruited to device surfaces beneath pneumatic valves, which protect surface-immobilized proteins from shear flows during reagent exchange.…”

Section: Advancing Existing Technologiesmentioning

confidence: 99%

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

et al. 2021

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High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants

Cited by 27 publications

References 108 publications

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

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