Monitoring cellular Guanosine triphosphate (GTP) and GTP associated proteins

Guanosine-5'-triphosphate (GTP) is an essential molecule for cell survival and function. Although free GTP plays a crucial role in several cellular processes, most studies focus solely on the protein bound GTP and its association with cancer. Efficient methods for determining cellular GTP concentration are currently lacking. Chromatography or capillary electrophoresis are commonly utilized for separation and mass spectrometry (MS) for quantification of GTP in biological samples. For monitoring GTPases and their interactions with ligands, differential scanning fluorimetry, differential scanning calorimetry, or differential static light scattering are typically utilized. However, these methods either lack sensitivity or throughput. In this PhD project, the Protein-Probe method, based on a Eu3+-chelate peptide probe that interacts with the hydrophobic core of the protein, has been developed and applied for studying different factors that affect the thermal stability of small GTPases, such as inhibitors, buffer ions and nucleotides. Furthermore, the method was explored for a better understanding of the binding specificity of the recent Food and Drug Administration approved KRASG12C covalent inhibitors to RAS GTPases. Additionally, a novel dual-labeled Förster Resonance Energy Transfer (FRET)-based peptide probe was introduced and compared to the Protein-Probe method in the thermal stability assay. The FRET-Probe technique was also applied to determine the chemical stability of proteins with different chemical denaturants. For measuring cellular GTP level, a homogenous high throughput assay was developed to measure the amount of GTP in cells utilizing a highly GTP-specific antibody. The assay yielded comparable results to those obtained with CE/MS, demonstrating a similar level of accuracy, while also exhibiting a substantial enhancement in throughput. In conclusion, this thesis work aimed at developing novel and robust methods to address the current limitations in monitoring cellular GTP concentration and to study small GTPases. The focus of the study was free GTP, recognizing its essential role in various cellular processes.------ Guanosiini-5'-trifosfaatti (GTP) on välttämätön molekyyli solujen selviytymiselle ja toiminnalle. Vaikka vapaalla GTP:llä on ratkaiseva rooli useissa soluprosesseissa, suurin osa tutkimuksista keskittyy vain GTPaaseihin sitoutuneeseen GTP:hen ja sen linkittymisessä syövän kehittymiseen. Tällä hetkellä solun GTP-konsentraation määrittämiseksi ei ole tehokkaita menetelmiä. Käyttämällä kromatografiaa tai kapillaarielektroforeesia (CE) erotukseen ja massaspektrometriaa (MS) kvantifiointiin voidaan biologisten näytteiden GTP-pitoisuutta seurata, mutta se on melko työlästä. GTPaasien ja niiden vuorovaikutusten seurantaan käytetään tyypillisesti differentiaalista pyyhkäisyfluorimetriaa, differentiaalista pyyhkäisykalorimetriaa tai differentiaalista staattista valonsirontaa. Kaikilta näistä menetelmistä puuttuu kuitenkin herkkyys tai suorituskyky. Tästä syystä tämän opinnäytetyön tavoitteena oli kehittää uusia herkkiä ja toimintavarmoja menetelmiä GTP-pitoisuuden seurantaan ja GTPaasien tutkimiseksi uudesta näkökulmasta. Solujen GTP-tasojen mittaamiseksi kehitettiin homogeeninen tehoseulontaan yhteensopiva määritys käyttämällä GTP-spesifistä vasta-ainetta. Määritys tuotti samanlaisia tuloksia kuin vertailumenetelmänä käytetty CE/MS, mutta huomattavasti nopeammin ja helpommin. Tässä väitöskirjatyössä esiteltiin myös uusi kaksoisleimattu FRET-pohjainen peptidikoetin, jota käytettiin yhdessä aiemmin kehitetyn ”Protein-Probe” menetelmän kanssa. FRET-Probe tarjoaa saman korkean herkkyyden kuin Protein-Probe tekniikka, mutta se mahdollistaa proteiinin stabiilisuuden mittaamisen neutraalissa pH:ssa ja yhdessä vaiheessa. Protein-Probe menetelmää käytettiin pienten GTPaasien ja niiden lämpöstabiilisuuteen vaikuttavien tekijöiden tutkimiseen. Lisäksi kehitettyjä menetelmiä käytettiin FDA:n äskettäin hyväksymien kovalenttisten KRAS(G12C) inhibiittorien sitoutumisspesifisyyden, toiminnan ja resistenttiyden muodostumismekanismien tutkimiseen. FRET-Probe-tekniikkaa ei sovellettu ainoastaan proteiinien lämpöstabiilisuuden seurantaan, vaan sen osoitettiin soveltuvan myös proteiinien isotermaalisen kemiallisen stabiilisuuden seurantaan

Nanoparticle-aided detection of colorectal cancer-associated glycoconjugates of extracellular vesicles in human serum

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.</p

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins

Protease substrate‐independent universal assay for monitoring digestion of native unmodified proteins

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.</p

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins

Rapid high-throughput compatible label-free virus particle quantification method based on time-resolved luminescence

Viruses play a major role in modern society and create risks from global pandemics and bioterrorism to challenges in agriculture. Virus infectivity assays and genome copy number determination methods are often used to obtain information on virus preparations used in diagnostics and vaccine development. However, these methods do not provide information on virus particle count. Current methods to measure the number of viral particles are often cumbersome and require highly purified virus preparations and expensive instrumentation. To tackle these problems, we developed a simple and cost-effective time-resolved luminescence-based method for virus particle quantification. This mix-and-measure technique is based on the recognition of the virus particles by an external Eu3+-peptide probe, providing results on virus count in minutes. The method enables the detection of non-enveloped and enveloped viruses, having over tenfold higher detectability for enveloped, dynamic range from 5E6 to 3E10 vp/mL, than non-enveloped viruses. Multiple non-enveloped and enveloped viruses were used to demonstrate the functionality and robustness of the Protein-Probe method

Droplet digital PCR for detection of mutations in high-grade serous ovarian cancer

Ovarian cancer (OC) is one of the most fatal cancers that affect female reproductive system. It is frequently discovered at advanced stage therefore, the 5-year survival rate is typically low. High grade serous ovarian cancer (HGSOC) is the most aggressive and has the worst prognosis of all types of OC. It is characterized by copy number variation (CNV) and genetic instability. Circulating free DNA (cfDNA) is a potential non-invasive alternative to tissue biopsy that can be used to detect mutations for early screening and targeted treatment of HGSOC. Nevertheless, methods with higher sensitivity than real-time PCR (qPCR) are required for amplification and quantification of plasma cfDNA. Droplet digital PCR (ddPCR) is a very sensitive tool in rare mutations detection. It produces absolute quantification without the need for a standard curve. Additionally, it divides the sample into droplets where each droplet acts as an individual PCR reaction. This leads to high sensitivity in identification rare targets. The aim of this study was to evaluate the sensitivity of ddPCR in detection of TP53 mutation and three novel unpublished fusion genes in HGSOC. For TP53 detection, serial dilutions of Mutant (MT) in genomic DNA (gDNA) was tested to identify limit of detection (LOD). Next, different concentrations of plasma cfDNA (5, 10, 20 and 40 ng per reaction) from HGSOC patient were analyzed by both qPCR and the optimized ddPCR assay. DdPCR showed 1 % LOD and recorded 3720 MT copies/ μl when 40 ng cfDNA per reaction was used. On the other hand, qPCR LOD was 5 % and it failed to detect cfDNA. Based on these results, we suggest that ddPCR is more sensitive than qPCR in detection of rare mutations. As for the fusion genes, ddPCR was more challenging and required further optimization

Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins

Beyond KRAS(G12C): biochemical and computational characterization of sotorasib and adagrasib binding specificity and the critical role of H95 and Y96

Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers, thus having been a target of intensive drug discovery efforts for decades. Lately, KRAS(G12C) switch-II pocket (SII-P)-targeting covalent small molecule inhibitors have finally reached the clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive non–small cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of the acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII-P binding by applying biochemical, cellular, and computational methods. Exact knowledge on SII-P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS- but not KRAS(G12C)-specific, due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound. Our results emphasize the accessibility of the SII-P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96