Characterization and therapeutic exploitation of molecular vulnerabilities in genetically defined lung cancer

Lung cancer is one of the most common cancer types and responsible for the largest number of cancer-related deaths worldwide. Typically, lung cancer arises in individuals with heavy smoking background and only rarely in never-smokers. Various cells of origin within the lung give rise to distinct, molecularly heterogenous lung cancer subtypes with the two major subtypes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Targeted therapy options also vary significantly between the specific subtypes and while oncogene-driven lung adenocarcinoma (LUAD) is already successfully treated with targeted drugs, no targeted therapies are available in SCLC. LUAD is often driven genetic alterations such as point mutations and rearrangements in genes of receptor tyrosine kinases (RTKs) like EGFR leading to aberrant activation of receptor tyrosine kinase signaling and oncogenic transformation. Mutation-selective small molecule RTK inhibitors have been developed to specifically kill oncogene-addicted cancer cells. Introduction of third generation EGFR inhibitor osimertinib substantially increased survival of EGFR-mutant LUAD patients but on-target resistance mutations such as EGFR G724S limit osimertinib efficacy leading to tumor relapse. Remarkably, we observed that second-generation EGFR inhibitor afatinib displayed selective activity against EGFR G724S in cell line and animal models. In contrast to osimertinib, afatinib still binds to EGFR G724S and reduces cellular viability, EGFR signaling, transformation and in vivo growth of EGFR G724S cells, therefore providing a possible treatment strategy for patients that relapse after osimertinib treatment due to EGFR G724S. Oncogenic gene fusions involving RET also lead to cellular transformation and LUAD tumorigenesis. Previously, multi-kinase inhibitors were used to treat RET-rearranged cancers with limited success due to lack of RET-specificity and RET gatekeeper mutations impeding inhibitor binding. We identified AD80, a type II kinase inhibitor that binds RET in the DFG-out conformation. AD80 displayed selective activity against common RET fusions KIFB-RET and CCDC6-RET and retained activity against RET V804M gatekeeper mutation. AD80 efficiently reduced RET- and downstream signaling as well as RET-associated gene expression. AD80 also displayed in vivo efficacy in CCDC6-RET patient-derived xenograft (PDX) models, demonstrating the potential of type II inhibitors as targeted therapy against RET-rearranged LUAD. In contrast to NSCLC, SCLC is defined by inactivation of tumor suppressors TP53 and RB1 and lacks targetable oncogenic drivers. Frequent activation of MYC transcription factor family members (MYC, MYCL, and MYCN) further accelerate tumor growth and aggressiveness. We found that activation of individual MYC family members entails differential molecular vulnerabilities. MYC overexpression is associated with high levels of DNA damage, repression of BCL2 expression and high apoptotic priming, leading to higher sensitivity towards Aurora kinase and MCL1 inhibition whereas high MYCL/MYCN expression is associated with resistance against these perturbations. Our study highlights that MYC status can be predictive for therapy response and might be used for molecularly-guided, patient stratification for future targeted therapy regimens in SCLC. A rare but very aggressive lung cancer type, NUT carcinoma is driven by BRD4-NUT fusion protein leading to large-scale epigenetic reprogramming and deregulated transcription of genes driving tumorigenesis. Using high-throughput viability screening, we identified that NUT carcinoma cells are preferentially sensitive against CDK9 inhibition. We observed, that CDK9 inhibition increases RNA Polymerase II pausing possibly reverting BRD4-NUT-mediated, transcriptional activation of pro-tumor genes warranting further investigation of CDK9 inhibition in NUT carcinoma

Application of the Nicoya OpenSPR to Studies of Biomolecular Binding: A Review of the Literature from 2016 to 2022

The Nicoya OpenSPR is a benchtop surface plasmon resonance (SPR) instrument. As with other optical biosensor instruments, it is suitable for the label-free interaction analysis of a diverse set of biomolecules, including proteins, peptides, antibodies, nucleic acids, lipids, viruses, and hormones/cytokines. Supported assays include affinity/kinetics characterization, concentration analysis, yes/no assessment of binding, competition studies, and epitope mapping. OpenSPR exploits localized SPR detection in a benchtop platform and can be connected with an autosampler (XT) to perform automated analysis over an extended time period. In this review article, we provide a comprehensive survey of the 200 peer-reviewed papers published between 2016 and 2022 that use the OpenSPR platform. We highlight the range of biomolecular analytes and interactions that have been investigated using the platform, provide an overview on the most common applications for the instrument, and point out some representative research that highlights the flexibility and utility of the instrument

Structural and biophysical characterization of novel GSK-3β inhibitors

The deregulation of GSK-3β (glycogen synthase kinase-3β) is involved in the pathogenesis of diverse diseases, such as cancer, diabetes and neurodegenerative disorders. Therefore, GSK-3β has become an attractive target for the design and development of new inhibitors for pathologies that present many limitations in therapeutic treatment. In the present thesis, we report a fast and efficient protocol for the expression of GSK-3β in insect cells with the baculovirus system, and employ purified GSK-3β in two different drug discovery projects. The first project describes the perspective validation of a novel computational method based on adiabatic bias molecular dynamics (ABMD) that aims to simulate protein-ligand unbinding events. Surface plasmon resonance (SPR) experiments performed on a series of GSK-3β inhibitors confirmed a coherence between predicted and experimental koff, highlighting the potential of this method for the calculation of residence times in hit-to-lead and lead optimization phases of drug discovery programs. Three novel X-ray crystal structures of pyrazine inhibitors in complex with GSK-3β are reported, providing further details in the binding mode of ATP-competitive GSK-3β inhibitors. The second project involves the structural and biophysical characterization of known GSK-3β allosteric inhibitors, and, in parallel, the discovery of novel allosteric modulators through computational and biophysical techniques. Herein, we describe the identification of promising molecules that have been selected by virtual screening and microscale thermophoresis (MST) analysis that display affinity to GSK-3β in the micromolar range. Taken together, our results provide useful insights for future rational drug design and discovery of small selective GSK-3β inhibitors

Biophysical analysis of protein-protein and protein-small molecule interactions

The validation of small molecule inhibitors identified by high throughput screening requires a set of robust assays for interaction analysis. Here I describe the implementation of three methods: bioluminescence resonance energy transfer (BRET) for the analysis of protein/protein interaction in cells, surface plasmon resonance (SPR) for the measurement of binding kinetics in vitro and capture compound mass spectrometry (CCMS) for the determination of binding specificity in proteome. All the methods described were tested on cytohesins, a family of guanine nucleotide exchange factors. The very recent discovery of their additional role in the regulation of receptor tyrosine kinases (RTKs) signalling and the availability of specific small molecule inhibitors (the Secins) made them an interesting target. BRET was applied to the analysis of a possible binding of the cytohesin ARNO to the RTK EGF receptor (EGFR). Two strategies were devised: a direct, where the interaction of ARNO and EGFR was monitored, and an indirect, which followed the changes in the EGFR/EGFR interaction upon overexpression of ARNO. Two SPR approaches were developed to analyse the interaction between ARNO and the EGFR on the one hand, and to determine the kinetic parameters of binding of ARNO to its inhibitor Secin16 on the other hand. For CCMS, a photoreactive affinity based SecinH3 probe (SecinH3-TPD) was synthesised and its ability to specifically label ARNO was shown, although the labelling yield was limited by low solubility. A protocol for the enrichment, digestion and MS-analysis of the labelled proteins was established

High Throughput Screening and mRNA-Display Selection for the Identification of Biologically Functional Molecules

Biologically functional molecules with desired properties have proven to be invaluable tools for investigating biological systems. Moreover, the identification of those functional molecules is a crucial task in modern drug discovery. These goals can be achieved by two major approaches: gscreeningh and gselectionh. Chapter 2 introduces one attractive method for gscreeningh to identify small molecule inhibitors of a given protein target from a small organic chemical library. The reverse transcriptase (RT) plays a crucial role in the early steps of the life cycle of HIV. Therefore HIV-1 RT serves as one of the major drug targets in anti-HIV therapy. Although current therapy for HIV-infected patients involving a combination of three or more of the following drugs: RT inhibitors, protease inhibitors, and/or viral entry inhibitors, has been highly successful, its long-term efficacy is severely limited by the emergence of drug-resistant variants of HIV. One way to address this problem is to identify new type of drugs with different inhibitory mechanisms to combat HIV. Thus, HIV-1 RT served a valuable target in the herein depicted screening approach. Protein-dependent alosteric ribozymes (or reporter ribozymes) are powerful tools which are able to sense molecular interactions, e.g. protein-protein, protein-nucleic acid, or protein-metabolite interactions in real-time. The expedience of this assay format makes it applicable to and suitable for high throughput screening in order to search for new molecules which disrupt such interactions. Reporter ribozyme FK-1 is a rationally engineered hammerhead ribozyme in a fusion construct with anti-RT aptamer, which can detect the presence of HIV-1 RT selectively in a domain specific manner. Thus, FK-1 enables to identify compounds via FRET-based fluorescence readout, which compete for the binding to HIV-1 RT with aptamer sequences inserted in the ribozyme construct. Through screening of 2500 small molecules using the reporter ribozyme (FK-1)-based assay, three potential inhibitors (28F6, 3E4 and 2E10) of HIV-1 RT were identified with in vitro inhibitory concentration that gave half-maximal activity (IC50) of 2-5 µM on DNA-dependent DNA polymerase activity. The three compounds were re-synthesized and further investigated to evaluate their selectivity and to elucidate the inhibitory mechanism. The selectivity was determined by testing inhibitory effect of the compound on other RTs (AMV and MMLV) and DNA polymerases from both prokaryotic (Klenow Fragment) and eukaryotic (human DNA polymerase ƒÀ) sources. Among the three compounds, 3E4 showed not only the lowest IC50 value for HIV-1 RT (2.1 µM) but the highest discrimination from all the other RT and DNA polymerases (10~50 fold) in terms of DNA-dependent DNA polymerase activity. The selectivity of 3E4 with the cognate RT from HIV-2 was a factor of approximately 4.5. In addition, the interaction between 3E4 and HIV-1 RT was observed by a surface plasmon resonance-based biosensor. Moreover, a cell-based assay for phenotypic analysis of sensitivity of HIV-1 to the three inhibitors using a self-inactivating virus vector system demonstrated that 3E4 inhibited marker gene expression in concentration dependent manner with a IC50 value of 5.3 µM. The effect of 3E4 was further confirmed with the similar reduction of HIV-1 replication and infectivity in vivo using a HIV wild type strain. In summary, the data strongly indicates that the small molecule 3E4 identified by reporter ribozyme-based screening is a highly specific and cellular active HIV-1 RT inhibitor, which has significant potentiality as novel type of anti-HIV drug. Furthermore, the approach using a reporter ribozyme assay could be broadly applicable as screening format. In chapter 3, the challenge of a gselectionh approach has been performed with the aim to evolve functional proteins from a random sequence protein library. Prior to the selection, the protein library consisting of a random 88 amino acid sequence was constructed by short cassettes ligations to avoid stop codons and frameshift mutations. The design of secondary structural patterns like ƒ¿-helix and ƒÀ-strand was introduced into the random regions. A display technology called mRNA-display was exploited to prepare protein library, where each protein sequence was covalently linked through its carboxy terminus to the 3f end of its encoding mRNA. Starting with a library that contained 1 ~ 1011 - 5 ~ 1012 different proteins, three in vitro selections were attempted to identify protein binders to small molecules such as cibacron blue 3GA and coenzymes (NADP and CoA). The selection against cibacron blue 3GA yielded enriched fractions retaining on the column matrix after the ninth cycle. These sequences could not be eluted by NAD, the structural analogue of cibacron blue 3GA, even at higher concentration. The small fractions eluted with NAD from this column have been cloned and sequenced, resulting in unexpectedly high frequency of frameshifts and internal stop codons. The alignment of full length intact sequences exhibited no conserved motif but the most appearance of ƒ¿ƒÀƒ¿ƒÀ type of cassette. The remaining column fractions were able to be eluted with free cibacron blue 3GA, implying those sequences might be specific cibacron blue binders. Other selections against both NADP and CoA did not enrich protein binders. The observed problems through the experiment concerning poor solubility and fold instability of mRNA-displayed proteins should be improved for further investigations

Xenobiotics interfering with corticosteroid action : from adrenal steroid synthesis to peripheral receptor activity

Corticosteroids are steroid hormones synthesized by the adrenal gland and regulating a variety of physiological processes to maintain whole-body homeostasis by acting through their corresponding receptors. Although the adrenal gland is considered one of the most toxin-vulnerable organs and steroid receptor regulation is recognized to have a considerable impact on tissue- and cell-specific steroid signaling, only few studies are currently exploring and characterizing the effects of xenobiotics on corticosteroid hormone action. The first part of this thesis aimed to establish optimized steroid profile analysis in cell culture supernatants and apply it in combination with further biological assessments and molecular modeling for the identification and characterization of exogenous chemicals potentially disrupting corticosteroid hormone production. A widely used in vitro model for studying effects of chemicals on adrenal steroid hormone synthesis constitutes the human H295R adrenocarcinoma cell line. Since the OECD test guideline No. 456 based on H295R cells has several limitations, this thesis refined the H295R steroidogenesis assay by simultaneously analyzing the most important adrenal steroid metabolites using a mass spectrometry-based method. A medium control at the beginning of the experiment as well as reference compounds with known mechanisms were introduced and, additionally, gene expression analyses were performed, in order to not only detect chemical-induced disturbances but also providing initial mechanistic insights into the mode-of-action of a given chemical. The newly established improved version of the H295R steroidogenesis assay was then further evolved by activating the cells either with torcetrapib, a potent inducer of corticosteroid synthesis, or with forskolin, a general inducer of steroidogenesis, allowing to assess the inhibitory potential of various test chemicals. The modified torcetrapib-stimulated H295R assay was then used to evaluate three selected hits from an in silico screening of environmental chemical databases using ligand-based pharmacophore models of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). This proof-of-concept for the application of pharmacophore-based virtual screening followed by biological assessment has proven suitable for assessing substances potentially interfering with corticosteroid synthesis. In another study within this thesis, the adapted version of the H295R steroidogenesis assay using forskolin-stimulated cells was applied to investigate the inhibitory effects of 19 anabolic androgenic steroids (AAS) and 3 selective androgen receptor modulators (SARMs). This enabled to group the test compounds according to their individual steroid patterns. Additionally, gene expression analysis, cell-free activity assays and molecular docking calculations contributed to providing initial mechanistic information. Besides direct effects on adrenal steroidogenesis, xenobiotic-induced alterations in circulating steroid hormone levels may arise due to altered feedback regulation or disturbed peripheral steroid metabolism. Thus, in a further part of this thesis drug-induced changes in steroid hormone levels were studied by measuring steroid profiles in human blood and urine samples. In a clinical study, plasma levels of steroid hormones and adrenocorticotropic hormone (ACTH) were analyzed in healthy volunteers administered a single dose of slow-release lisdexamfetamine (100 mg) or immediate-release D-amphetamine (40.3 mg) at equimolar doses. Importantly, lisdexamfetamine and D-amphetamine similarly enhanced the levels of glucocorticoids, androgen precursors and ACTH, suggesting an acute stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Although lisdexamfetamine showed a delayed time of increase and peak levels of plasma D-amphetamine concentrations compared to the D-amphetamine treatment, drug exposure and drug effects seemed to be comparable between the two formulations. In a clinical case study, a comprehensive analysis of blood and urinary steroid profiles was conducted in samples from two patients receiving posaconazole, an antifungal agent associated with hypertension and hypokalemia due to mineralocorticoid excess. Steroid analyses indicated interindividual differences in the mechanism of mineralocorticoid-based hypertension with preferential CYP11B inhibition in one patient and predominant inhibition of 11β-HSD2 in the second patient. These results show that steroid profiling in plasma and urine samples can not only reveal disturbances of steroid homeostasis but also provide initial mechanistic information. Together, these findings emphasize that molecular modeling combined with biological evaluation represents a valuable approach for the identification and characterization of chemicals potentially interfering with corticosteroid production and to provide initial mechanistic insights. However, in vivo investigations are unavoidable to study the impact of chemicals acting on the HPA axis. Xenobiotics may not only affect steroid hormone production, feedback regulation or pre-receptor control of corticosteroid metabolism, but may also interfere directly with the receptor and steroid signal transduction. In order to understand potential disturbances of glucocorticoid action by xenobiotics, it is important to further clarify the signaling pathways involved in glucocorticoid receptor (GR) activation. Therefore, another part of this thesis focused on the impact of the serine/threonine-specific protein phosphatase PP1α on the activity of the GR. PP1α was found to increase GR activity, and preliminary mechanistic investigations showed that levels of phosphorylated GR-Ser211 were altered and glycogen synthase kinase 3 might be involved. Hence, PP1α appeared to modulate the cellular response to glucocorticoids, implying that impairment of its activity could lead to aberrant glucocorticoid hormone action. In conclusion, these studies identified a novel GR regulating protein that enhances cortisol stimulation by controlling GR phosphorylation. A profound understanding of glucocorticoid signaling might provide the basis for developing cell models and conditions for the detection of chemicals disturbing glucocorticoid sensitivity and thereby contributing to diseases

Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

BACKGROUND: HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1. RESULTS: Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9\u27s Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1. CONCLUSION: We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs

Bio-functionalised nanoparticles for enzyme sensing

Inorganic nanoparticles and their accompanying diverse physical properties are now virtually in routine use as imaging tools in cell-biology. In addition to serving as excellent contrast agents, their size- and environment-dependent optical and magnetic properties can be harnessed to create enzyme biosensor devices of extremely high sensitivity, whilst circumventing the numerous technical limitations associated with traditional enzyme assays. This thesis explores new applications of noble metal and semiconductor nanoparticles (quantum dots) for the detection of a range of medically-relevant enzymes. Here, water soluble colloidal gold nanoparticles and quantum dots are modified with peptides and antibodies to afford new reagents for enzyme sensing. These bio-functionalised nanoparticle probes exhibit numerous advantages over traditional enzyme sensing systems owing to their robust and size-tuneable optical properties. These enzyme-responsive nanoparticle systems are tailored to detect the activity of protein kinase and histone acetyltransferase activity in a simple homogeneous assay format based on Förster resonance energy transfer (FRET). Furthermore, these systems can be applied to screen for putative small-molecule modulators of enzyme function. These new assays should provide the basis for the development of a variety of new enzyme detection strategies based on nanoparticlespecific optical phenomena

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, “hyper-pluripotent” state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency