AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Fabrication and Characterization of Micromachined Active Probes With Polymer Membranes for Biomolecular Force Spectroscopy

A micromachined polymer membrane-based active probe has been developed for biomolecular force spectroscopy. The probe has integrated but significantly decoupled electrostatic actuation and optical interferometric force sensing capabilities. Devices have been fabricated on silicon substrates using Parylene as the membrane material. The electrostatic actuator integrated into the probe could provide > 1-μm displacement with a flat response of up to 30 kHz in fluid, a feature particularly useful in fast-pulling force spectroscopy experiments involving biomolecules. The probes were successfully employed to measure the unbinding forces between biotin and streptavidin, wherein the force noise level was <;10 pN with a 1-kHz bandwidth for an 8-N/m membrane with a 25-kHz resonance frequency in fluid. This is in agreement with the thermal noise data generated by a finite-element model that predicts further improvements with simple design changes

A micromachined membrane-based active probe for biomolecular mechanics measurement

A novel micromachined, membrane-based probe has been developed and fabricated as assays to enable parallel measurements. Each probe in the array can be individually actuated, and the membrane displacement can be measured with high resolution using an integrated diffraction-based optical interferometer. To illustrate its application in single-molecule mechanics experiments, this membrane probe was used to measure unbinding forces between L-selectin reconstituted in a polymer-cushioned lipid bilayer on the probe membrane and an antibody adsorbed on an atomic force microscope cantilever. Piconewton range forces between single pairs of interacting molecules were measured from the cantilever bending while using the membrane probe as an actuator. The integrated diffraction-based optical interferometer of the probe was demonstrated to have <10 fm Hz−1/2 noise floor for frequencies as low as 3 Hz with a differential readout scheme. With soft probe membranes, this low noise level would be suitable for direct force measurements without the need for a cantilever. Furthermore, the probe membranes were shown to have 0.5 µm actuation range with a flat response up to 100 kHz, enabling measurements at fast speeds

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for \u3e30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation

Microtubules growing and shortening under constant force

&lt;div class="abstract"&gt; &lt;div class="abstract-content selected"&gt; &lt;p&gt;Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses &lt;em&gt;in vitro&lt;/em&gt;. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for &gt;30 min, providing a close match to the persistent coupling seen&lt;em&gt; in vivo&lt;/em&gt; between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.&lt;/p&gt; &lt;/div&gt; &lt;/div&gt;&lt;p&gt;Funding provided by: National Institutes of Health&lt;br&gt;Crossref Funder Registry ID: https://ror.org/01cwqze88&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;Funding provided by: Howard Hughes Medical Institute&lt;br&gt;Crossref Funder Registry ID: https://ror.org/006w34k90&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;In these experiments, microtubules were grown with a yeast-kinetochore-decorated bead attached to the microtubule's plus end. We applied constant forces ranging from 0.5 to 17 pN to the plus end via the bead and recorded the microtubule's growth and/or shortening, as well as the stochastic switching between these two states.&lt;/p&gt

Microtubules growing and shortening under constant force

&lt;div class="abstract"&gt; &lt;div class="abstract-content selected"&gt; &lt;p&gt;Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses &lt;em&gt;in vitro&lt;/em&gt;. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for &gt;30 min, providing a close match to the persistent coupling seen&lt;em&gt; in vivo&lt;/em&gt; between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.&lt;/p&gt; &lt;/div&gt; &lt;/div&gt;&lt;p&gt;Funding provided by: National Institutes of Health&lt;br&gt;Crossref Funder Registry ID: https://ror.org/01cwqze88&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;Funding provided by: Howard Hughes Medical Institute&lt;br&gt;Crossref Funder Registry ID: https://ror.org/006w34k90&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;In these experiments, microtubules were grown with a yeast-kinetochore-decorated bead attached to the microtubule's plus end. We applied constant forces ranging from 0.5 to 17 pN to the plus end via the bead and recorded the microtubule's growth and/or shortening, as well as the stochastic switching between these two states.&lt;/p&gt