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

Insights into the homeostatic regulation of I kappa B alpha

The regulation of the transcription factor NF-$$kappa$$B is crucial to proper cell physiology, as misregulation of this transcription factor can lead to many disease states, including chronic inflammation and cancer. NF-$$kappa$$B is inhibited by a class of inhibitor proteins known as I kappa B; the most effective I$$kappa$$B is I$$kappa$$B$$alpha$$. Signal induced degradation of I$$kappa$$B$$alpha$$ leading to NF-$$kappa$$B translocation and activation is well documented and requires post-translational modifications such as phosphorylation and ubiquitination. It has recently been demonstrated that I$$kappa$$B alpha also undergoes stimulus-independent degradation and this degradation pathway might be important for NF-$$kappa$$B activity regulation. The focus of this study is to investigate the mechanism of stimulus independent degradation of I$$kappa$$B alpha and its effect on NF- $$kappa$$B activity. Chapter 1 introduces the NF-$$kappa$$B: I$$kappa$$B signaling system, ubiquitin-independent degradation of several substrates, and also various regulatory proteasome complexes. Chapter 3 describes the delineation of the pathways regulating the degradation of I$$kappa$$B$$alpha$$. Results presented here show that the degradation pathway of I$$kappa$$B alpha is determined by binding to NF-$$kappa$$B subunits. It is further shown that perturbations of ubiquitin-independent degradation pathway alter NF-$$kappa$$B activation. Chapter 4 focuses on the ankyrin repeat sequence of I$$kappa$$B alpha. Of the six ankyrin repeats present in I$$kappa$$B alpha, several deviate from the consensus ankyrin repeat sequence. Mutations back to the consensus sequence in several ankyrin repeats demonstrate that the location of thermodynamic stabilization determines the degradation rate of I$$kappa$$B$$alpha$$. Finally, Chapter 5 dissects the degradation requirements of I$$kappa$$B alpha. Our results show that there are two degrons within I$$kappa$$B$$alpha$$; one located in the 5th ankyrin repeat, and the other within the PEST domain of I$$kappa$$B$$alpha$$. Both degrons are controlled by hydrophobic residues within long stretches of flexible regions

Insights into the homeostatic regulation of I kappa B alpha

The regulation of the transcription factor NF-$$kappa$$B is crucial to proper cell physiology, as misregulation of this transcription factor can lead to many disease states, including chronic inflammation and cancer. NF-$$kappa$$B is inhibited by a class of inhibitor proteins known as I kappa B; the most effective I$$kappa$$B is I$$kappa$$B$$alpha$$. Signal induced degradation of I$$kappa$$B$$alpha$$ leading to NF-$$kappa$$B translocation and activation is well documented and requires post-translational modifications such as phosphorylation and ubiquitination. It has recently been demonstrated that I$$kappa$$B alpha also undergoes stimulus-independent degradation and this degradation pathway might be important for NF-$$kappa$$B activity regulation. The focus of this study is to investigate the mechanism of stimulus independent degradation of I$$kappa$$B alpha and its effect on NF- $$kappa$$B activity. Chapter 1 introduces the NF-$$kappa$$B: I$$kappa$$B signaling system, ubiquitin-independent degradation of several substrates, and also various regulatory proteasome complexes. Chapter 3 describes the delineation of the pathways regulating the degradation of I$$kappa$$B$$alpha$$. Results presented here show that the degradation pathway of I$$kappa$$B alpha is determined by binding to NF-$$kappa$$B subunits. It is further shown that perturbations of ubiquitin-independent degradation pathway alter NF-$$kappa$$B activation. Chapter 4 focuses on the ankyrin repeat sequence of I$$kappa$$B alpha. Of the six ankyrin repeats present in I$$kappa$$B alpha, several deviate from the consensus ankyrin repeat sequence. Mutations back to the consensus sequence in several ankyrin repeats demonstrate that the location of thermodynamic stabilization determines the degradation rate of I$$kappa$$B$$alpha$$. Finally, Chapter 5 dissects the degradation requirements of I$$kappa$$B alpha. Our results show that there are two degrons within I$$kappa$$B$$alpha$$; one located in the 5th ankyrin repeat, and the other within the PEST domain of I$$kappa$$B$$alpha$$. Both degrons are controlled by hydrophobic residues within long stretches of flexible regions

Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans

The EphA3 G518L lung cancer mutation enhances <i>cis</i> interaction with ephrin-A3.

<p>(A) HEK AD-293 cells were infected with a lentivirus encoding mCherry-ephrin-A3 or mCherry as a control. The cells were then transfected with EphA3 ΔN or the EphA3 ΔN G518L mutant. EphA3 immunoprecipitates were probed with an anti-ephrinA3 antiserum and reprobed for EphA3. The EphA3 G518L mutation found in lung cancer increases the affinity of the lateral interaction between EphA3 and ephrin-A3. The histogram shows normalized means ± SE quantified from the immunoblots from 3 experiments. *p<0.05 by one sample t test for the comparison of the EphA3 ΔN G518 mutant versus EphA3 ΔN. (B) A549 lung cancer cells were infected with a lentivirus encoding EphA3 wild-type or the G518L mutant and ZsGreen alone or together with a lentivirus encoding mCherry-ephrin-A3; control cells were infected with lentiviruses encoding ZsGreen and mCherry. The histogram shows cell binding of ephrin-A3 AP (one experiment) and ephrin-A5 AP (2 experiments), confirming that ephrin-A3 coexpression prevents the binding of ephrin AP proteins to the EphA3 G518L mutant. Normalized means from 3 experiments (each with duplicate samples) ± SE are shown. ***p<0.001 by one-way ANOVA and Tukey’s post-hoc test for the comparison of cells coexpressing EphA3 and ephrin-A3 with cells only expressing EphA3 and for the comparison of cells coexpressing EphA3 G518L and ephrin-A3 with cells only expressing EphA3 G518L. The immunoblot of the cell lysates shows expression of EphA3, ephrin-A3, and β-tubulin as loading control.</p

Coexpressed ephrin-B2 attenuates EphB4 as well as EphA3 activation in cancer cells.

<p>(A) The histogram shows the binding of ephrin-B2 AP to A549 cells infected with lentiviruses encoding EGFP-ephrin-B2 or EGFP, revealing that ephrin-B2 coexpression inhibits ephrin-B2 AP binding to EphB4. Normalized means from 3 experiments (each with triplicate samples) ± SE are shown. ***p<0.001 by unpaired t test for the comparison of cells expressing ephrin-B2 with cells not expressing ephrin-B2. The immunoblot of the cell lysates shows expression of EphB4, ephrin-B2 and β-tubulin as loading control. (B) A549 lung cancer cells and MCF7 breast cancer cells were infected with lentiviruses encoding EGFP-ephrin-B2 or EGFP. EphB4 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphB4. Cell lysates were probed for ephrin-B2 with an anti-EGFP antibody and for β-tubulin as loading control. The histograms show normalized means ± SE quantified from 2 immunoblots for each cell line. *p<0.05 by one sample t test for the comparison of ephrin-B2 Fc-treated cells expressing ephrin-B2 with cells not expressing ephrin-B2. (C) A549 cells were infected with a lentivirus encoding EphA3 and ZsGreen together with a lentivirus encoding EGFP-ephrin-B2 or EGFP only. Control cells were infected with lentiviruses encoding ZsGreen and EGFP. EphA3 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA3. Lysates were probed for ephrin-B2 with an anti-EGFP antibody as well as for EphA3 and for β-tubulin as loading control. The histogram shows normalized means ± SE quantified from 2 immunoblots. *p<0.05 by one sample t test for the comparison of ephrin-A3 Fc-treated cells expressing ephrin-B2 with cells not expressing ephrin-B2. (D) Ephrin-A5 AP binding to cell surface EphA3 is inhibited by ephrin-B2 coexpression. The histogram shows means ± SE from 3 experiments (each with triplicate samples) for the binding of ephrin-A5 AP or ephrin-B2 AP to the A549 cells used for the experiment in C. For ephrin-A5 binding, **p<0.01 by one-way ANOVA and Dunnett’s post-hoc test for the comparison with cells expressing EphA3 and EGFP; for ephrin-B2 AP binding, **p<0.01 by unpaired t test for the comparison of cells expressing or not expressing EphA3. The immunoblot of the cell lysates shows expression of ephrin-B2, EphA3 and β-tubulin as loading control, verifying that ephrin-B2 coexpression did not reduce EphA3 levels. Of note, the doublet corresponding to overexpressed ephrin-B2 is not due to different degrees of N-linked glycosylation because removal of N-linked oligosaccharides with the PNGase-F endoglycosidase similarly increased the SDS-PAGE mobility of both bands (not shown). Whether the upper band may represent a form with O-linked oligosaccharides [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#B51" target="_blank">51</a>] or other posttranslational modification remains to be determined.</p

<i>Cis</i> interaction between coexpressed EphA3 and ephrin-A3 does not require the regions involved in trans interaction.

<p>(A) HEK AD-293 cells were infected with a lentivirus encoding mCherry-ephrin-A3 or mCherry as a control. Subsequently, the cells were transfected with plasmids encoding full-length EphA3 or a truncated form lacking the ligand-binding domain and cysteine-rich region (EphA3 ΔN). EphA3 immunoprecipitates were probed with anti-ephrin-A3 antiserum and reprobed for EphA3, revealing that ephrin-A3 association with EphA3 does not require the EphA3 ligand-binding domain. The histogram shows normalized means ± SE quantified from the immunoblots from 2 experiments. p>0.05 by one sample t test for the comparison of ephrin-A3 bound to EphA3 ΔN or full-length EphA3. (B) HEK AD-293 cells infected with a lentivirus encoding mCherry-ephrin-A3, the mCherry-ephrin-A3 E129K mutant, or mCherry as a control, were transfected with a plasmid encoding EphA3 ΔN. EphA3 immunoprecipitates were probed for ephrin-A3 and reprobed for EphA3, revealing that the E129K mutation does not abolish the <i>cis</i> interaction with EphA3. The histogram shows normalized means ± SE quantified from 3 immunoblots. p>0.05 by one sample t test for the comparison of ephrin-A3 E129K versus ephrin-A3 wild-type bound to EphA3 ΔN. (C) HEK AD-293 cells were transfected with control pcDNA3, pcDNA3-ephrin-A3, or pcDNA3-ephrin-A3 E129K. The histogram shows means from two experiments for the binding of EphA3 AP to ephrin-A3, confirming that ephrin-A3 E129K mutant does not bind EphA3 in <i>trans</i>. ***p<0.001 by one-way ANOVA and Dunnett’s post-hoc test for the comparison with cells expressing wild-type ephrin-A3. The immunoblot shows the expression of ephrin-A3 and ephrinA3 E129K in lanes loaded with equal amounts of total lysates. It should be noted that ephrin-A3 overexpressed in HEK cells yields two bands, with the upper band corresponding to the size of the mature full-length protein.</p

Removal of endogenous ephrin-As from the cell surface potentiates EphA2 activation by soluble ephrin-A1 in <i>trans</i>.

<p>(A) SKBR3 and (B) MCF7 breast cancer cells were treated with PI-PLC for 4 hours and then stimulated with ephrin-A1 Fc. EphA2 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA2. Lysates probed with anti-ephrin-A1 antibody verify removal of ephrin-As by PI-PLC; β-tubulin verifies equal loading of the lanes. The Odyssey LI-COR system was used for detection and the color images were converted to greyscale with Photoshop. The histograms show the normalized data from 3 different experiments *p<0.05 and ***p<0.001 by one sample t test for the comparison of ephrin-A1 Fc-stimulated cells treated or not with PI-PLC. (C) SKBR3 cells were treated with PI-PLC as in A or with the broad-spectrum matrix metalloprotease inhibitor GM-6001 for 24 hours. Immunoprecipitates and lysates were probed as indicated.</p

Coexpressed ephrin-A3 attenuates EphA receptor activation in cancer cells.

<p>(A,B) NCI-H226 and A549 lung cancer cells were infected with a lentivirus encoding EphA3 and ZsGreen alone or together with a lentivirus encoding mCherry-ephrin-A3; control cells were infected with lentiviruses encoding ZsGreen and mCherry. EphA3 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA3. Lysates were probed for mCherry-ephrin-A3 with an anti-dsRed antibody that also recognizes mCherry, for EphA3, and for β-tubulin as loading control. The histograms show normalized means ± SE quantified from 3 immunoblots in both A and B. In one of the A549 experiments used for quantification, the cells were stimulated with ephrin-A5 Fc. **p<0.01 by one sample t test for the comparison of ephrin-A3 Fc-treated cells expressing both EphA3 and ephrin-A3 with ephrin-A3 Fc-treated cells expressing only EphA3. Of note, EphA3 levels were higher in A549 cells co-expressing ephrin-A3/ephrin-B2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g002" target="_blank">Figs. 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g003" target="_blank">3B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g004" target="_blank">4A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081445#pone-0081445-g005" target="_blank">5C,D</a>), suggesting that this receptor may be stabilized by the coexpressed ephrins. (C) A549 cells were infected with a lentivirus encoding mCherry-ephrin-A3 or mCherry as a control. Immunoprecipitated endogenous EphA2 was probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA2. Lysates were probed with an anti-dsRed antibody and β-tubulin as loading control. The histogram shows normalized means ± SE quantified from 3 immunoblots. **p<0.01 by one sample t test for the comparison of ephrin-A3 Fc-treated cells expressing or not expressing ephrin-A3.</p