Photoluminescence quenchers in drug discovery

During the drug discovery process molecular libraries containing millions of molecules are screened to find a single potential drug with the highest potency and efficacy over unwanted side-effects. The whole drug development process usually takes more than a decade and requires billions of euros. Thus, new methods with improved cost and time effectiveness are vital for both improving primary screening of molecular libraries and verifying the results obtained via secondary screens. Drug discovery often utilizes methods based on photoluminescence, the emission of light from a luminophore molecule excited by photon absorption, e.g. fluorescence. The methods monitor changes in photoluminescence that are initiated by an event in the assay e.g. interaction of protein and ligand. In some methods, the detectability of the change can be enhanced by decreasing the luminescence of noninteracting luminophores by quenching. Some luminophores are quenched by water but others require dedicated quencher molecules e.g. the acceptor molecules quenching donor luminophores in Förster resonance energy transfer (FRET). In the work presented in this thesis, the addition of a soluble quencher was utilized for developing two photoluminescence-based assays suitable for drug discovery for studying protein-ligand interactions (PLI), protein-protein interactions (PPI), and protease activity. The first method developed, QTR-FRET, utilizes soluble quencher to decrease the emission of non-interacting donor luminophore thus preventing it from interfering in the monitored acceptor channel. QTR-FRET improves conventional TR-FRET by permitting the investigation of low affinity PLI interactions requiring high donor luminophore concentrations and by enabling multiplexing via monitoring PLI and subsequent PPI. The second method, the Protein-Probe, was developed to measure the emission of external luminescence probe during its interaction with a target protein. The emission of non-interacting probe is reduced by soluble quencher. The Protein-Probe is suitable for monitoring the PLIs via their effect on the thermal stability of protein and the protease activity via the digested protein substrate. The Protein-Probe has 50-fold improved sensitivity compared to conventional methods.Loistevalon sammuttajat lääkekehityksessä Varhaisen lääkekehityksen aikana miljoonia molekyylejä sisältävistä molekyylikirjastoista seulotaan lääkemolekyyli, joka vaikuttaa sairauteen mahdollisimman tehokkaasti välttäen haitallisia sivuvaikutuksia. Yleensä lääkekehitysprosessi kestää yli vuosikymmenen ja kuluttaa miljardeja euroja. Tämän vuoksi uusia aika- ja kustannustehokkaita menetelmiä tarvitaan parantamaan ensisijaista seulontaa sekä varmistamaan niiden tulokset toissijaisella seulonnalla. Varhaisessa lääkekehityksessä hyödynnetään usein menetelmiä, jotka perustuvat fotoluminesenssiin, eli valon tuotantoon luminofori molekyylistä, joka on virittynyt fotonin imeytymisen vuoksi, kuten fluoresenssiin. Menetelmät tarkkailevat luminesenssiin vaihtelua, minkä saa aikaan tapahtuma määrityksessä kuten proteiinin ja ligandin vuorovaikutus. Joissain menetelmissä tapahtuman havaitsemista voidaan tehostaa sammuttamalla sitoutumattomien luminoforien luminesenssia. Jotkut luminoforit voidaan sammuttaa vedellä, mutta toiset vaativat erillisen molekyylin kuten Förster resonanssi energiasiirron (FRET) luovuttaja luminoforeja sammuttavat vastaanottaja molekyylit. Tässä väitöskirjassa liukoisen sammuttajan lisäystä käytettiin kehittämään kaksi lääkekehitykseen soveltuvaa fotoluminesenssimenetelmää, jotka tarkkailevat proteiini-ligandi (PLV), proteiini-proteiini vuorovaikutuksia sekä proteaasien aktiivisuutta. Ensimmäiseksi kehitetyssä QTR-FRET menetelmässä sammuttajaa käytetään vähentämään sitoutumattomien luovuttaja-luminoforien valon tuotantoa, jolloin niiden aiheuttama häiriö vastaanottajamolekyylin luminesenssia mitattaessa vähenee. QTR-FRET mahdollistaa korkeaa luovuttajapitoisuutta tarvitsevien matalan affiniteetin PLV:en sekä kahden linkittyneen vuorovaikutuksen tarkkailun yhteisestä kaivosta. Toinen kehitetty menetelmä, Protein-Probe, mittaa erillisen koettimen luminesenssia sen sitoutuessa proteiiniin. Sitoutumattoman koettimen luminesenssi himmentyy liukoisen sammuttajan vaikutuksesta. Protein-Probella voi tarkkailla PLV:ia proteiinien lämpöpysyvyyden välityksellä, sekä proteaasien aktiivisuutta niiden hajottamien substraattiproteiinien kautta. Protein-Probe menetelmän herkkyys on 50-kertainen verrattuna vastaaviin menetelmiin

Parechovirusinfektioiden laukaisema luontainen immuunipuolustus soluviljelmissä

Ihmisen parechovirukset (HPeV:t) voivat aiheuttaa vakavia keskushermostoinfektioita vastasyntyneille. Tyypin 3 HPeV on yleisin aiheuttaja, mutta Tauriaisen ryhmä löysi Suomesta 2012 lapsia, joille HPeV4 aiheutti vakavan yleisinfektion. Sitä, miten HPeV:t aiheuttavat hermostoinfektioita, ei tunneta. Tarkoituksena on tutkia, miten sytokiinien ilmeneminen infektoiduissa soluissa eroaa eri virustyyppien ja -alatyyppien välillä. Tutkimme tätä infektoimalla ihmisen keuhkoepiteelisoluja (A549) sekä astrosyyttisoluja (T98G) ihmisen parechoviruksilla: HPeV1-Harris, HPeV3, HPeV4-FI ja HPeV4-NL sekä kontrollina Coxsakievirus B3:lla (CV-B3). Osa viruksista oli aiheuttanut vakavia tauteja ja osa lieviä. Käytimme kontrollina myös kaksinauhaista RNA:ta (Poly-IC), joka laukaisee sytokiinien tuotannon. Infektoidut solut ja niiden kasvatusliemi kerättiin 24 h, 48 h ja 72 h aikapisteissä. Määritimme solujen ilmentämiä sytokiineja kasvatusliemistä Proteome Profiler -kitillä. Tulosten perusteella valitsimme jatkotutkimusten sytokiinit. Niiden tuotantoa mitattiin kvantitatiivisella RT-PCR analyysillä sekä immunofluoresenssi värjäyksellä. Kaikkien virusalatyyppien sytokiiniprofiilit vaihtelivat keskenään ja solulinjojen välillä. HPeV3:n ja CV-B3:n välillä nähtiin samankaltaisuutta. Suurimmat erot nähtiin interleukiini- 6:n (IL-6) tuotannossa: kaikki virukset estivät sen tuottoa A549 soluissa mutta tehostivat sitä T98G soluissa. Tutkimus on edelleen käynnissä, joten tulokset ovat alustavia. Tulokset viittaavat siihen, että HPeV:n sytokiiniprofiilit muistuttavat toisiaan ja CV-B3:n sytokiiniprofiileja. Kaikkien virusten sytokiiniprofiilit erosivat joiltain osin toisistaan, mutta vakavaa sairautta aiheuttaneiden virusten sytokiiniprofiilit eivät eronneet huomattavasti muiden virusten profiileista

Sensitive, homogeneous, and label-free protein-probe assay for antibody aggregation and thermal stability studies

Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu3+-conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60 degrees C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques

QTR-FRET: Efficient background reduction technology in time-resolved förster resonance energy transfer assays

A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Forster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Forster resonance energy transfer (TR-FRET) was developed to reduce background signal in the conventional energy transfer applications. The TR-FRET functionality is often limited by the lanthanide donor background signal leading to the use of low donor concentration. QTR-FRET reduces this background by introducing soluble quencher molecule, and in this work the concept functionality was proven and compared to previously introduced QRET and TR-FRET technologies. Comparison was performed with three different Eu3+-chelates exhibiting different luminescent lifetime and stability. The side-by-side comparison of the three signaling systems and Eu3+ -chelates was demonstrated in a model assay with Eu3+-chelate conjugated biotin and streptavidin (SA) or Cy5-SA conjugate. Comparison of the methodologies showed increased signal-to-background ratios when comparing QTR-FRET to TR-FRET, especially at high Eu3+ -biotin concentrations. Quenching the non-bound Eu3+-biotin improved the assay performance, which suggests that an improved assay performance can be attained with the QTR-FRET method. QTR-FRET is expected to be especially useful for Eu3+-labeled ligands with low affinity or assays requiring high Eu3+-ligand concentration. The QTR-FRET indicated potential for multi-analyte approaches separately utilizing the direct QRET-type Eu3+-chelate signal and energy transfer signal readout in a single- well. This potential was hypothesized with Avi-KRAS nucleotide exchange assay as a second biologically relevant model system

Sensitive Label-Free Thermal Stability Assay for Protein Denaturation and Protein-Ligand Interaction Studies

In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein-ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies

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

Nanomolar Protein-Protein Interaction monitoring with a label-free protein-probe technique

Protein-protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Förster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody

Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets

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

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