2,237 research outputs found
De novo drug design through artificial intelligence: an introduction
Developing new drugs is a complex and formidable challenge, intensified by rapidly evolving global health needs. De novo drug design is a promising strategy to accelerate and refine this process. The recent introduction of Generative Artificial Intelligence (AI) algorithms has brought new attention to the field and catalyzed a paradigm shift, allowing rapid and semi-automatic design and optimization of drug-like molecules. This review explores the impact of de novo drug design, highlighting both traditional methodologies and the recently introduced generative algorithms, as well as the promising development of Active Learning (AL). It places special emphasis on their application in oncological drug development, where the need for novel therapeutic agents is urgent. The potential integration of these AI technologies with established computational and experimental methods heralds a new era in the rapid development of innovative drugs. Despite the promising developments and notable successes, these technologies are not without limitations, which require careful consideration and further advancement. This review, intended for professionals across related disciplines, provides a comprehensive introduction to AI-driven de novo drug design of small organic molecules. It aims to offer a clear understanding of the current state and future prospects of these innovative techniques in drug discovery
Limitations of Protein Structure Prediction Algorithms in Therapeutic Protein Development
The three-dimensional protein structure is pivotal in comprehending biological phenomena. It directly governs protein function and hence aids in drug discovery. The development of protein prediction algorithms, such as AlphaFold2, ESMFold, and trRosetta, has given much hope in expediting protein-based therapeutic discovery. Though no study has reported a conclusive application of these algorithms, the efforts continue with much optimism. We intended to test the application of these algorithms in rank-ordering therapeutic proteins for their instability during the pre-translational modification stages, as may be predicted according to the confidence of the structure predicted by these algorithms. The selected molecules were based on a harmonized category of licensed therapeutic proteins; out of the 204 licensed products, 188 that were not conjugated were chosen for analysis, resulting in a lack of correlation between the confidence scores and structural or protein properties. It is crucial to note here that the predictive accuracy of these algorithms is contingent upon the presence of the known structure of the protein in the accessible database. Consequently, our conclusion emphasizes that these algorithms primarily replicate information derived from existing structures. While our findings caution against relying on these algorithms for drug discovery purposes, we acknowledge the need for a nuanced interpretation. Considering their limitations and recognizing that their utility may be constrained to scenarios where known structures are available is important. Hence, caution is advised when applying these algorithms to characterize various attributes of therapeutic proteins without the support of adequate structural information. It is worth noting that the two main algorithms, AlfphaFold2 and ESMFold, also showed a 72% correlation in their scores, pointing to similar limitations. While much progress has been made in computational sciences, the Levinthal paradox remains unsolved
Signaling Mechanisms Behind the Benefits of Sleep
Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende Würmer nach einer Verletzung häufiger. Insgesamt deutet dies darauf hin, dass AMP als Signalmoleküle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. Für diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlüsseln, durch den verschiedene Reize der angeborenen Immunität, das heißt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche Domänen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-Domäne als aktive Proteasedomäne von NAS-38 angesehen wird, erwartete ich eine Schlüsselrolle dieser Domäne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitätsinduziertem Schlaf? Da gezeigt wurde, dass AMP während des NAS-38 Schlafes und auch nach Verwundung hochreguliert sind, erwartete ich, dass AMP an der Signalisierung von Schlaf von der Epidermis zum Nervensystem beteiligt sind. In einem zweiten Schritt untersuchte ich die molekularen Mechanismen, die den Vorteilen von Schlaf für das Überleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: Verändert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf für viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeinträchtigt. Zweitens, ist Schlaf wichtig für die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? Während der Larvenentwicklung fällt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeinträchtigt. Methoden: Zur Analyse der Signalmechanismen, durch die sowohl NAS-38 als auch Verletzungen Schlaf induzieren, filmte ich das Schlafverhalten von C. elegans mittels Langzeit-Bildgebung in Agarose-Mikrokammern. So führte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 Domäne deletiert war. Darüber hinaus testete ich verschiedene Suppressoren für immunvermittelten Schlaf, der durch NAS 38 oder Verletzungen induziert war. Die Redundanz des Suppressionseffektes der verschiedenen Mitglieder der AMP-Familie auf immunvermittelten Schlaf testete ich, indem ich den Suppressionsphänotyp einer CRISPR/Cas9-editierten Multi-Knockout-Mutante analysierte, in der insgesamt 19 AMP deletiert waren. Um Effektoren zu identifizieren, die den AMP nachgeschaltet sind, induzierte ich Schlaf durch Überexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschäftigte ich mich mit der Frage, wie genau Schlaf das Überleben nach Verletzungen unterstützt. Ich verglich die Expression von literaturbekannten Reportern für verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell für chronisch-genetischen Schlafmangel. Darüber hinaus habe ich die Transkriptome zwischen jeweils adulten verwundeten und unverwundeten Wildtypen und schlaflosen Mutanten verglichen. Um die Struktur der Cuticula des Wildtyps und der schlaflosen Mutante zu vergleichen, analysierte ich außerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-Domäne verlängert. Dieser Prozess wird moderiert durch die TSP-1-Domäne. Weiterhin konnte ich zeigen, dass viele AMP redundant wirken um immunvermittelten Schlaf, verursacht durch NAS-38 oder Verletzungen, zu signalisieren. Ich konnte zeigen, dass das AMP NLP-29 über den Neuropeptidrezeptor NPR-12 wirkt. Dieser kann NLP-29-induzierten Schlaf vermitteln, wenn er in einem neuronalen Netzwerk exprimiert wird, welches nachweislich RIS aktiviert. Interessanterweise fand ich außerdem heraus, dass für NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verändert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verändert. Dies betraf unter anderem eine Gruppe oszillierender Gene, die Cuticula-assoziierte Proteine codieren, und deren Expression normalerweise ihren Höhepunkt gegen Ende des Lethargus erreicht. Da angenommen wird, dass der Zeitpunkt der Kollagenexpression entscheidend für eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsächlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die Strapazierfähigkeit der Cuticula gegenüber bestimmten Belastungen verringern. Daher könnte Schlaf erforderlich sein, Robustheit in Form einer strukturierten Cuticula zu fördern. Schlussfolgerungen: In diesem Dissertationsprojekt vollendete ich die Charakterisierung eines neuentdeckten Mechanismus in C. elegans, durch den Verwundungen Schlaf als Teil der Immunantwort aus der Peripherie zum Nervensystem signalisieren. Ich konnte zeigen, dass AMP gewebeübergreifend Signale von der Epidermis an ein neuronales Netz vermitteln, welches wiederum RIS aktiviert und dadurch Schlaf induziert. Da Komponenten dieses Signalweges konserviert sind, könnten AMP auch in anderen Tieren, einschließlich des Menschen, Schlaf zur Genesung fördern. Darüber hinaus habe ich die Grundlagen für die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes für Heilung und Überleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verändern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfältigere Rolle bei der zeitlichen Regulierung der Genexpression hin.:Summary I
Zusammenfassung IV
Contents VII
List of Figures XII
List of Tables XIV
Abbreviations XV
1. Introduction 1
1.1. Sleep is fascinating 1
1.1.1. The origin and basic features of sleep 1
1.1.2. Regulation of sleep in higher animals 3
1.1.2.1. Neuronal control of sleep 3
1.1.2.2. Molecular control of sleep 5
1.1.3. The functions of sleep 6
1.2. The immune system and its relationship to sleep 7
1.3. Wound healing and its relationship to sleep 10
1.4. Caenorhabditis elegans is a well-studied model organism 12
1.4.1. Sleep in C. elegans 15
1.4.2. The C. elegans cuticle 18
1.4.3. Immunity in C. elegans 19
1.4.4. Wound healing response in C. elegans 22
2. Previous results 25
2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25
2.2. NAS-38 increases sleep mostly through the RIS neuron 25
2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25
2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27
2.5. Overexpression of AMPs induces RIS dependent quiescence 30
2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31
3. Thesis Aims 34
3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34
3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35
4. Materials and Methods 36
4.1. C. elegans maintenance 36
4.2. C. elegans crossing and genotyping 41
4.3. Creation of transgenic animals 45
4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45
4.3.2. Microparticle bombardment 45
4.3.3. CRISPR/Cas9 system 46
4.4. Synchronizing worm cultures by hypochlorite treatment 48
4.5. Imaging 49
4.5.1. Imaging setups 49
4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50
4.5.2.1. Imaging of heterozygous mutants 50
4.5.3. DIC imaging in the temperature control device 51
4.5.4. Fluorescent imaging experiments 51
4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51
4.5.4.2. nlp-29p::GFP in L4 larvae 52
4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52
4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52
4.5.4.5. Functional Ca2+ imaging in young adults 52
4.5.4.6. Fluorescence imaging across the whole developmental time 54
4.5.4.7. Nuclear decompaction assays 55
4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55
4.5.4.9. Imaging DPY-13::mKate2 in young adults 56
4.6. Image analysis 56
4.6.1. Assessment of developmental time and lethargus detection 56
4.6.2. Sleep detection in DIC mode 56
4.6.3. Analyzing functional Ca2+ images 57
4.6.4. Fluorescent reporter analysis during long-term imaging 57
4.7. RNAi-by-feeding 58
4.8. Transcriptome analysis 59
4.8.1. Analysis of the nas-38(ok3407) transcriptome 59
4.8.2. Analysis of the wounding transcriptome 59
4.9. Epidermal wounding 62
4.9.1. Laser wounding 62
4.9.2. Needle wounding 62
4.9.3. Survival assay 63
4.10. Scanning Electron Microscopy (SEM) 63
4.11. Histamine-inducible hyperpolarization of RIS 64
4.12. Cuticle integrity test with Sodium hypochlorite 64
4.13. NPR-12 receptor modeling 64
4.14. Quantification and statistical analysis 65
5. Results 66
5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66
5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66
5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66
5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69
5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72
5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75
5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77
5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80
5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81
5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83
5.1.10. AMPs contribute to the survival after wounding 85
5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87
5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87
5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87
5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89
5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95
5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101
5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106
5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109
5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114
5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120
5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122
6. Discussion and Outlook 128
6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128
6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130
6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131
6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132
6.5. Genetic sleep deprivation alters cuticle formation 135
6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137
6.7. Conclusion 139
7. References 140
8. Acknowledgements 163
9. Appendix 166
9.1. Standard reagents 166
9.2. Sequence summary of PHX3754 167
9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171
9.4. Permissions to reprint figures 174
9.5. Experimental author contributions 175
9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176
9.7. Overlap of the adult wounding transcriptome with other data sets 179
9.8. Curriculum Vitae – Marina Patricia Sinner 181Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a protective response. This hypothesis, however, was hitherto lacking final evidence and pathway components.
Research questions and hypotheses: I aimed to characterize the molecular mechanism by which separate triggers of innate immunity, i. e. NAS-38 and wounding, induce sleep. I specifically addressed two questions: Firstly, which domains of the NAS-38 protein are involved in sleep regulation? As the astacin domain is predicted to be the active protease domain of NAS-38, I expected a role for it also in sleep induction by NAS-38. Secondly, what is the role of AMPs in signaling immunity-induced sleep? As they have been shown to be upregulated during times of increased sleep in the nas-38 mutant and after wounding, I expected AMPs to be involved in signaling sleep from the epidermis to the nervous system. In a second step, I investigated the molecular mechanisms underlying the benefits of sleep for surviving injury. Again, I addressed two questions: Firstly, does genetic sleep deprivation alter the transcriptional wounding response? As sleep has a role in many fundamental processes and sleeplessness increases mortality upon wounding, I hypothesized that genetic sleep deprivation impairs wounding-induced changes of transcriptional activity. Secondly, does sleep help building robustness before encountering injury? During larval development the synthesis of a new cuticle coincides with sleep. Thus, I hypothesized that genetic sleep deprivation impairs proper cuticle formation. Methods: To dissect the signaling mechanisms by which NAS-38 and wounding induced sleep, I followed sleep behavior of C. elegans by long-term imaging in agarose microchambers. I performed a structure-function analysis with different nas-38 mutants, each carrying a deletion of a different domain. Moreover, I screened for suppressors of sleep induced by NAS 38 or wounding. To test for redundancy of the AMP family, I investigated the suppression-phenotype of a CRISPR/Cas9 edited multi-knockout mutant lacking 19 AMPs. To identify downstream effectors of the AMP NLP 29, I induced sleep by overexpressing NLP 29 from a heat-shock promoter and analyzed the suppression-phenotype of different knockout mutants. For the second project, I addressed the question how sleep aids recovery from injury. I followed fluorescent reporters of previously described wounding response pathways by fluorescent long-term imaging in wild-type and genetically sleep-deprived animals. Moreover, I compared the transcriptomes of adult wild-type and genetically sleep-deprived worms both wounded and unwounded. To investigate the structure of the cuticle, I analyzed scanning electron microscopy images. Results: In the first project, I could show that NAS-38 indeed increases sleep via its astacin domain in a process that is modulated by the TSP-1 domain. Moreover, I could show that many AMPs act redundantly in mediating immunity-induced sleep downstream of NAS-38 and after wounding. I demonstrated that the AMP NLP-29 signals sleep via the neuropeptide receptor NPR 12. This receptor can mediate sleep when it is specifically expressed in command interneurons of a circuit that has been shown to activate RIS. Interestingly, I also found that EGFR signaling is required to mediate NLP-29-induced sleep.
In the second project, I found that sleeplessness does not dramatically alter the transcriptional wounding response. However, I could show that transcription is altered already in the unwounded non-sleeping mutant. This affects, among others, a specific subset of oscillating collagen-coding genes, whose expression usually peaks around the end of lethargus. As the timing of expression of collagens is thought to be highly important for proper cuticle formation, I characterized the cuticle of the aptf-1(gk794) mutant. I could show that young adult aptf 1(gk794) worms indeed have a structural defect affecting cuticular furrows in the region adjacent to the alae, which could potentially decrease specific aspects of resilience of the cuticle. Thus, sleep might be required to build robustness in the form of a properly structured cuticle. Conclusion: In this PhD project, I completed the characterization of a novel mechanism by which wounding signals sleep from the periphery to the nervous system as part of the immune response in C. elegans. I could show that AMPs act as cross-tissue signals from the epidermis to a neuronal RIS-controlling circuit that ultimately leads to sleep induction. As components of this molecular pathway are highly conserved, AMPs might also induce sleep to promote recovery from injury in other organisms, including humans. Moreover, I laid the foundations for dissecting the molecular mechanisms behind the functions of sleep for healing and survival. Even though the disability to sleep did not seem to drastically change the transcriptional response to wounding, my results indicate a role for
Out-of-Distribution Generalization of Deep Learning to Illuminate Dark Protein Functional Space
Dark protein illumination is a fundamental challenge in drug discovery where majority human proteins are understudied, i.e. with only known protein sequence but no known small molecule binder. It\u27s a major road block to enable drug discovery paradigm shift from single-targeted which looks to identify a single target and design drug to regulate the single target to multi-targeted in a Systems Pharmacology perspective. Diseases such as Alzheimer\u27s and Opioid-Use-Disorder plaguing millions of patients call for effective multi-targeted approach involving dark proteins. Using limited protein data to predict dark protein property requires deep learning systems with OOD generalization capacity. Out-of-Distribution (OOD) generalization is a problem hindering the application and adoption of deep learning in real world problems. Classic deep learning setting in contrast is assuming training and testing data are independent identically distributed (iid). A well trained model under iid setting with reported 98% accuracy could deteriorate to worse than random guess in deployment to OOD data significantly different from training data. Numerous techniques in the research field emerged but are only addressing some specific OOD scenario instead of a general one. Dark protein illumination has unique complexity comparing to common deep learning tasks. There are three OOD axes, protein-OOD, compound-OOD, interaction-OOD. Previous research have only focused on compound-OOD, where new compound design algorithms are developed but still for 500 common proteins, instead of whole human genome 20,000 proteins, and only for single-targeted paradigm instead of multi-targeted. Focusing on an instrumental problem in drug discovery, dark protein function illumination problem is introduced from the OOD perspective. A series of dark protein OOD algorithms are developed to predict dark protein ligand interaction where multiple instrumental deep learning techniques are adapted to the biology context. By proposing the dark protein illumination problem, highlighting the neglected axes, demonstrating possibilities, numerous diseases now embrace new hopes
Identification of biomarkers of the retinoic acid signaling pathway in the zebrafish embryo model to predict human developmental toxicants
This dissertation falls within the context of the paradigm shift in regulatory toxicology testing which promotes using a mechanistic-based approach based on in vitro tests instead of traditional animal testing to predict chemical hazards to human such as developmental toxicity. The novel research expands the understanding of developmental toxicity pathways by studying chemically-induced gene expression changes related to the perturbation of the retinoic acid signaling pathway (RA-SP) in a vertebrate embryo model. By using the zebrafish embryo (ZE) model it was possible to take advantage of the conservation of this biological pathway across vertebrate taxa, to predict potential human developmental toxicity. The ZE model is not new, however it has been primarily used and optimized for its morphology readout due to the transparent eggs enabling morphological observations during chemical exposure. However, improvements and harmonization are necessary to utilize this model with a reliable molecular level readout, to reveal relevant changes in gene expression. In chapter 2, the protocol design was refined to identify gene expression (GE) changes in the ZE. This was done by investigating the optimal exposure duration to study such changes due to the perturbation of the RA-SP. An exposure of ZE to the RA-SP agonist all-trans retinoic acid (ATRA) was performed using 6 different exposure durations, ranging from 2-117 hrs. These results identified that 4h exposure was the optimal exposure duration to study chemically-induced GE regulation specifically related to the RA-SP perturbation, thereby optimizing the ZE protocol for GE analysis. In chapters 3 and 4, the optimized ZE-GE protocol was employed to identify GE biomarker candidates for maldevelopment. After exposing ZE to two teratogenic compounds known to perturb the RA-SP (ATRA and Valproic Acid, VPA) and one non-teratogenic control compound (Folic Acid, FA), the chemically-induced perturbation of the RA-SP was explored using a whole genome scale GE analysis approach (RNAseq). The 3 test compounds each showed a specific mRNA expression profile, with 248 genes commonly regulated by both teratogenic compounds (ATRA and VPA) but not by FA. These 248 genes were implicated in several developmental processes. 62 differentially expressed genes (DEGs) were associated with nervous system development and were further examined in Chapter 3. These 62 genes were identified as potential biomarkers of early neurodevelopmental toxicity. In chapter 4, the perturbation of RA-SP on the GE associated with development of mesoderm derived tissues was investigated using bioinformatics methods. The investigation identified gene ontology (GO)-terms related to 47 DEGs. Literature indicates that these genes were normally expressed among 3 mesodermal sections (paraxial, intermediate, and lateral plate section) and 6 mesodermal tissues (somites, striated muscle, bone, kidney, circulatory system, and blood). These 47 DEGs were identified as potential biomarkers of early mesodermal maldevelopment or novel potential biomarkers for specific mesodermal organs. These proposed biomarker candidates advance the knowledge on the retinoic acid-mediated developmental toxicity mechanism. As their responses become even more broadly characterized by exploring different exposure regimes and the profiles of different chemicals, these biomarkers could contribute to predictive tools in animal-free chemical hazard and risk assessment
Computational Approaches to Drug Profiling and Drug-Protein Interactions
Despite substantial increases in R&D spending within the pharmaceutical industry, denovo drug design has become a time-consuming endeavour. High attrition rates led to a
long period of stagnation in drug approvals. Due to the extreme costs associated with
introducing a drug to the market, locating and understanding the reasons for clinical failure
is key to future productivity. As part of this PhD, three main contributions were made in
this respect. First, the web platform, LigNFam enables users to interactively explore
similarity relationships between ‘drug like’ molecules and the proteins they bind. Secondly,
two deep-learning-based binding site comparison tools were developed, competing with
the state-of-the-art over benchmark datasets. The models have the ability to predict offtarget interactions and potential candidates for target-based drug repurposing. Finally, the
open-source ScaffoldGraph software was presented for the analysis of hierarchical scaffold
relationships and has already been used in multiple projects, including integration into a
virtual screening pipeline to increase the tractability of ultra-large screening experiments.
Together, and with existing tools, the contributions made will aid in the understanding of
drug-protein relationships, particularly in the fields of off-target prediction and drug
repurposing, helping to design better drugs faster
In-silico assay of a dosing vehicle based on chitosan-TiO2 and modified benzofuran-isatin molecules against Pseudomonas aeruginosa
A high priority of the World Health Organization (WHO) is the study of drugs against Pseudomonas aeruginosa, which has developed antibiotic resistance. In this order, recent research is analyzing biomaterials and metal oxide nanoparticles, such as chitosan (QT) and TiO2 (NT), which can transport molecules with biological activity against bacteria, to propose them as drug carrier candidates. In the present work, 10 modified benzofuran-isatin molecules were studied through computational simulation using density functional theory (DFT) and molecular docking assays against Hfq and LpxC (proteins of P. aeruginosa). The results show that the ligand efficiency of commercial drugs C-CP and C-AZI against Hfq is low compared with the best-designed molecule MOL-A. However, we highlight that the influence of NT promotes a better interaction of some molecules, where MOL-E generates a better interaction by 0.219 kcal/mol when NT is introduced in Hfq, forming the system Hfq-NT (Target-NT). Similar behavior is observed in the LpxC target, in which MOL-J is better at 0.072 kcal/mol. Finally, two pharmacophoric models for Hfq and LpxC implicate hydrophobic and aromatic-hydrophobic fragments
A computational view on nanomaterial intrinsic and extrinsic features for nanosafety and sustainability
In recent years, an increasing number of diverse Engineered Nano-Materials (ENMs), such as nanoparticles and nanotubes, have been included in many technological applications and consumer products. The desirable and unique properties of ENMs are accompanied by potential hazards whose impacts are difficult to predict either qualitatively or in a quantitative and predictive manner.
Alongside established methods for experimental and computational characterisation, physics-based modelling tools like molecular dynamics are increasingly considered in Safe and Sustainability-by-design (SSbD) strategies that put user health and environmental impact at the centre of the design and development of new products. Hence, the further development of such tools can support safe and
sustainable innovation and its regulation.
This paper stems from a community effort and presents the outcome of a four-year-long discussion on the benefits, capabilities and limitations of adopting physics-based modelling for computing suitable features of nanomaterials that can be used for toxicity assessment of nanomaterials in combination with data-based models and experimental assessment of toxicity endpoints. We review modern multiscale physics-based models that generate advanced system-dependent (intrinsic) or timeand
environment-dependent (extrinsic) descriptors/features of ENMs (primarily, but not limited to nanoparticles, NPs), with the former being related to the bare NPs and the latter to their dynamic fingerprinting upon entering biological media. The focus is on (i) effectively representing all nanoparticle attributes for multicomponent nanomaterials, (ii) generation and inclusion of intrinsic nanoform properties, (iii) inclusion of selected extrinsic properties, (iv) the necessity of considering distributions of structural advanced features rather than only averages. This review enables us to identify and highlight a number of key challenges associated with ENMs’ data generation, curation,
representation and use within machine learning or other advanced data-driven models to ultimately enhance toxicity assessment. Finally, the set up of dedicated databases as well as the development of grouping and read-across strategies based on the mode of action of ENMs using omics methods are identified as emerging methodologies for safety assessment and reduction of animal testing
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