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

Intégration et caractérisation de couches sensibles de ZnO:Ga et de cobaltites spinelles (CoxFe3-xO4 ; 1≤x≤3) sur véhicules de test pour la détection de dioxyde d'azote sub-ppm et d'éthanol.

National audienceMeasuring air pollutants is a challenge. Most gaseous pollutants, such as nitrogen dioxide (NO2) or volatile organic compounds (VOCs), occur at parts per billion (ppb) levels in the air and are mixed with thousands of other compounds. Today, the public air quality monitoring network relies on fixed measuring stations, monitoring organisations use the most efficient but expensive detectors and the cost of a multi-detector station is high, thus the spatial resolution of the measurements is strongly reduced.In this thesis we propose to find materials capable of detecting very low concentrations of polluting gases and to integrate them by a vapour deposition technique in the form of thin layers (a few tens of nanometres thick) on micro-platforms in order to manufacture low-cost resistive gas sensors. To this end, gallium-doped zinc oxide (ZnO:Ga) was first studied for the detection of NO2. The latter was deposited by radio frequency sputtering on fused silica substrates for structural and microstructural characterisation. The resulting layer appears as a columnar and circular polycrystalline growth with a porous surface, ideal for gas adsorption. X-ray diffraction shows a wurtzite structure with the (002) plane parallel to the substrate. The electrical and thermoelectric characterisations show a low resistive n-type material (compared to bulk ZnO). These ZnO:Ga thin films were deposited on characterisation platforms (developed and fabricated during this thesis) especially for these preliminary detection tests under controlled atmosphere, but also for the determination of the optimal detection parameters of the material. These tests proved that at 250°C the ZnO:Ga has a good sensitivity to 500 ppb NO2 in an atmosphere with 50% relative humidity. Then, the thin films were deposited on micro-platforms using a shadowmask (developed and fabricated for this process) to obtain well localised deposits in order to fabricate the sensor and characterise it under real operating conditions. Thus, the fabricated sensors are very sensitive to 500 ppb NO2 at 50% relative humidity. In parallel to the study of ZnO:Ga, an exploratory evaluation was conducted on iron cobalt powders (denoted CoxFe3-xO4 with 1≤x≤ 3) for the detection of VOCs. Powders with different compositions were studied: Co1.16Fe1.84O4, Co1.5Fe1.5O4, Co1.7Fe1.3O4, Co2FeO4, Co2.7Fe0.3O4 and Co3O4. These powders were deposited on alumina substrates heated to different temperatures and tested under 25 ppm ethanol in dry air. Of all the compositions, Co3O4 showed the best response at low temperatures.In conclusion, this thesis presents ZnO:Ga thin films as a promising material for NO2 detection at very low concentrations. However, it remains to determine a more correct way to exploit this material, depending on the applications, taking into account its assets (sensitivity, response time, temperature ...) and its shortcomings (aging etc...). Co3O4 seems to be interesting for ethanol detection, and it is now necessary to study its thin layers in order to detect lower concentrations and to integrate it into micro-platforms for the manufacture of functional sensorsLa mesure des polluants atmosphériques est un défi. La plupart des polluants gazeux, tels que le dioxyde d'azote (NO2) ou les composés organiques volatile (COV), se produisent à des niveaux de parties par milliard (part per billion ou ppb) dans l'air et sont mélangés avec des milliers d'autres composés. Aujourd'hui, le réseau public de surveillance de la qualité de l'air repose sur des stations de mesure fixes, les organismes de contrôle utilisent les détecteurs les plus performants, mais les plus onéreux et le coût d'une station multi-détection étant élevé, la résolution spatiale des mesures se trouvent fortement réduites.Dans le cadre de cette thèse nous proposons de trouver des matériaux capables de détecter de très faibles concentrations de gaz polluants et de les intégrer par une technique de dépôt en phase vapeur sous forme de couche minces (de quelques dizaines de nanomètre d’épaisseur) sur des micro-plateformes dans le but de fabriquer des capteurs de gaz résistifs low-cost. Dans cette objectif, l’oxyde de zinc dopé au gallium (noté ZnO : Ga) a tout d’abord été étudié pour la détection du NO2. Ce dernier a été déposé par pulvérisation cathodique par radiofréquence sur des substrats de silice fondu pour des caractérisations structurale et microstructurales. La couche obtenue se présente comme une croissance colonnaire et circulaire polycristallin avec une surface poreuse, idéale pour l’adsorption des gaz. La diffraction aux rayons X montre une structure wurtzite avec le plan (002) parallèle au substrat. Les caractérisations électriques et thermoélectriques montrent un matériau de type-n, peu résistif (comparé au ZnO brut). Ces couches minces de ZnO:Ga ont été déposé sur des plateformes de caractérisation (développé et fabriqué durant cette thèse) spécialement pour ces essais préliminaires de détection sous atmosphère contrôlée, mais aussi pour la détermination des paramètres de détection optimales du matériau. Ces essais ont prouvé qu’à 250°C le ZnO :Ga présente une bonne sensibilité à 500 ppb de NO2 dans une atmosphère comportant 50% d’humidité relative . Ensuite, les couches minces ont été déposé sur des micro-plateformes en utilisant un shadowmask (mis au point et fabriqué pour ce procédé) pour obtenir des dépôts bien localisé afin de fabriquer le capteur et de le caractériser dans des conditions de fonctionnement réelles. Ainsi, les capteurs fabriqués sont très sensibles à 500 ppb de NO2 avec 50 % d’humidité relative. Parallèlement à l’étude de ZnO : Ga, une évaluation exploratoire a été mené sur des poudres de cobaltites de fer (noté CoxFe3-xO4 avec 1≤x≤ 3) pour la détection des COVs. Des poudres avec différentes compositions ont été étudié : Co1.16Fe1.84O4, Co1.5Fe1.5O4, Co1.7Fe1.3O4, Co2FeO4, Co2.7Fe0.3O4 et Co3O4. Ces poudres ont été déposé sur des substrats d’alumine porté à différentes températures et testé sous 25 ppm d’éthanol sous air sec. Parmi toutes les compositions, le Co3O4 présente la meilleure réponse à basse température.En conclusion, cette thèse présente les couches minces de ZnO :Ga comme étant un matériau prometteur pour la détection du NO2 à très faible concentration. Il reste cependant à déterminer une façon plus correcte d’exploiter ce matériau, dépendant des applications, en prenant en compte ses atouts (sensibilité, temps de réponses, température …) et ses défauts (vieillissement etc…). Le Co3O4 semble intéressant dans la détection de l’éthanol, il faut à présent porter l’étude sur les couches minces de celui-ci pour détecter des concentrations plus basses et l’intégrer sur les micro-plateformes pour fabriquer les capteurs fonctionnels

Human papillomaviruses activate and recruit SMC1 cohesin proteins for the differentiation-dependent life cycle through association with CTCF insulators.

Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses

Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle

Human papillomaviruses (HPVs) are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR) to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6–directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV) replication underscoring the importance of this cellular transcription factor in the life cycles of multiple human cancer viruses

CTCF consensus motifs are found in HPV genomes.

<p>A). Schematic diagram of linear HPV 31 genome showing the location of the CTCF (CCCTC) consensus motifs that are also present in other high-risk HPV subtypes. Number indicates the location of the beginning of the pentamer sequence. B). Summary of analysis of 125 HPV types for the presence of CTCF core consensus motifs in L2, L1, E2 and E4 regions. Percentage of HPV type that are positive for these sequences is shown. (C,D). Chromatin immunoprecipitation analysis of SMC1 and γ-H2AX binding to URR region in undifferentiated cells and cells differentiated in calcium for 72 hours. (E,F) Chromatin immunoprecipitation analysis of SMC1 and γ-H2AX binding to the L2 region in undifferentiated cells and cells differentiated in high calcium media for 72 hours. SMC1 binds to the L2 region at similar levels in both undifferentiated or differentiated cells. γ-H2AX binding to HPV genomes increases upon differentiation at both URR and L2 regions Quantitative real-time PCR was performed using a Lightcycler 480 (Roche). Similar results were seen in three independent experiments.</p

KLF4 binds to the Upstream Regulatory Region (URR) of HPV-31 and activates viral promoter reporter constructs.

<p>(3A). HPV-31 Upstream Regulatory Region is represented as a linear schematic with potential transcription factor binding sites marked in black rectangles. KLF4 consensus binding regions: region1 (R1) and region2 (R2) are marked as patterned black rectangles. (3B). The two KLF4 binding regions in the viral URR were analyzed for KLF4 binding in CIN-612 cells using Chromatin Immunoprecipitation (ChIP) assay. Binding of KLF4 to both URR regions was significantly enriched over IgG controls in both undifferentiated (UD) and differentiated (D) conditions (3B). p values: *<0.01, **<0.001. (3C). Single nucleotide changes were introduced into the whole HPV-31 genome, HFKs transfected with wildtype and mutant genomes and stable cell lines selected. The stably transfected cells were designated as R1M (region 1 of URR) and R2M (region 2 of URR). (i) Both R1M and R2M cells displayed impaired viral DNA amplification upon differentiation compared to the wild type cells as shown by Southern blot analysis. (ii) Both mutants produced significantly reduced late transcripts upon differentiation compared to the wild type cells as shown in the northern blot. (3D). KLF4 expression plasmid was co-transfected with either URR- or Lpro- (late promoter) luciferase reporter plasmids into 293T cells, and relative luciferase activities were measured. KLF4 activated luciferase activity of both URR and Lpro constructs at both low and high concentrations.</p

Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle

<div><p>Human papillomaviruses (HPVs) are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR) to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6–directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including <i>TCHHL1</i>, <i>VIM</i>, <i>ACTN1</i>, and <i>POT1</i>, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV) replication underscoring the importance of this cellular transcription factor in the life cycles of multiple human cancer viruses.</p></div

Knockdown of SMC1 with shRNA blocks viral genome amplification.

<p>A). CIN612 were infected with lentiviruses encoding shRNAs to SMC1 and after 48 hours total cell extracts were isolated and analyzed by Western blot for levels of SMC1. Quantitation of band intensity revealed an approximate 50% knockdown of SMC1 protein levels on average across three experiments. The knockdown is statistically significant between mock and shSMC and shCTRL and shSMC, p≤.0001, and p≤.001 respectively. B). shRNA knockdown of SMC1 blocks HPV-31 differentiation-dependent viral amplification. Southern blot analysis of CIN 612 cells infected with lentiviruses encoding shRNAs to SMC1 and induced to differentiate in methylcellulose for 48 hours. UD stands for undifferentiated while D stands for differentiated. Total DNA was isolated and examined by Southern analysis for HPV amplification. Similar results were seen in four independent experiments using two different shRNAs as well as the combination of the two. Quantification of band intensity was determined by densitometry using Image J software and represents an average of three independent experiments. SMC knockdown results in a reduction in amplification that is statistically significant, p≤.05</p

SMC1 is phosphorylated by both ATM and ATR.

<p>A). ATM inhibitor, KU60019 inhibits CHK2 phosphorylation, but only partially reduces SMC1 phosphorylation in HPV positive cells. HPV 31 positive CIN 612 cells were treated with the ATM kinase inhibitor KU60019 (10uM) in DMSO or DMSO alone for 48 and 72 hours and cell extracts examined by Western blot analysis. pCHK2 levels are inhibited by KU60019 while pSMC1 levels are only partially reduced suggesting another kinase such as ATR is involved in phosphorylation. GAPDH is included as a loading control. B) DNA damage response inhibitor Caffeine (10mM) inhibits both pSMC1 and pATR after 48 hours of treatment.</p

Mutation of 3 CTCF core consensus sequences within the L2 coding region of the HPV 31 genome results in impaired growth, loss of episomes, and rapid integration into the host genome.

<p>A).HFKs were transfected with WT HPV-31 genomes or HPV-31 genomes harboring mutations within three CTCF motifs in the L2 coding region along with a drug selectable marker. Following transfection cells were selected and representative colony sizes as seen by light microscopy are shown three days after initiation of selection. Differences in colony size were reproducibly seen. B). Mutation of CTCF sites impairs genome maintenance resulting in integration and inability to amplify. Following transfection and selection, cells were grown to confluency and differentiated in high calcium media for 72 hours. Total DNA was isolated and examined by Southern blot analysis for HPV amplification. Quantification of band intensity was determined by densitometry using FIJI software. C). Mutation of 3 CTCF core consensus sequences abrogates SMC1 and CTCF binding to the HPV genome. Chromatin Immunoprecipitation analysis of stable cell lines containing the WT HPV-31 genome and the three mutations in CTCF motifs demonstrated a reduction of SMC and CTCF binding in mutant lines. Two separate passages of the mutant cell lines demonstrates loss of SMC-1 and CTCF binding. Data is representative of three independent experiments. Panel i). L2 region in cells with wildtype HPV 31; ii). L2 region in cells with L2 mutant 3X genomes at passage 2 after transfection; iii). L2 region in cells with L2 mutant 3X genomes at passage 3 after transfection.</p