Peptide-based drug discovery tools for protein post-translational modifications

Bringing a new drug to the market is an expensive and long process. Typically, more than ten years and a billion dollars needs to be spent so the whole process is a big investment for a pharma company. The hit generation phase of the drug development is one of the first steps of the development and there, millions of potential molecules are screened against a known druggable target in order to generate hits, i.e., molecules that have a specific activity towards the target. The hit generation phase requires high throughput screening (HTS) assays that are capable of screening millions of molecules rapidly with low expenses with platforms that are robust. This thesis project focused on developing assays for hit generation phase for targets that catalyse protein post-translational modifications (PTMs). PTMs are regulatory mechanisms of cells that control many cellular events and are therefore involved in many disease conditions, for example, cancer. PTMs are additions or cleavages of chemical groups, typically catalysed by enzymes which activity can be affected with inhibitor or activator molecules. Today, the techniques available for the activity monitoring of PTM-catalysing enzymes suffer from many weaknesses. Many methods, such as mass spectrometry or heterogeneous luminescence-based techniques, are not applicable for HTS, which increases the screening times of molecule libraries and therefore the cost of screening. Alternatively, some techniques are developed for one of a few different PTMs and therefore requiring a new platform for each PTM. On top of these, many methods are expensive to perform due to low sensitivity or antibodies, making the development of new assays motivating. The work presented in this thesis expanded the Peptide-Break technology to be more well suited in PTM-detection and developed one new assay to detect cysteinespecific PTMs. The developed assays are simple homogeneous techniques applicable for HTS providing new interesting options for inhibitor screening

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

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

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

Uusien proteiinin lämpödenaturaatioon perustuvien lääkeaineseulontamenetelmien kehittäminen

Lääkeaineseulonnassa uusille entistä herkemmille ja monikäyttöisimmille menetelmille on jatkuva tarve. Lämpödenaturaatioon perustuvien luminesenssimenetelmien vahvuutena on niiden suurempi herkkyys suhteessa huoneenlämpötilassa tehtyyn analyysiin yhdistettynä luminesenssidetektorin pieneen määritysrajaan. Lämpödenaturaatiomenetelmien periaatteena on saada kohdeproteiini denaturoitumaan, jotta siihen sitoutuvan lääkeainemolekyylin vaikutus siihen voidaan havaita esimerkiksi denaturaatiolämpötilan nousuna. Kokeellisen osan tavoitteena oli kehittää aikaerotteiseen luminesenssiin perustuvia menetelmiä, joita voitaisiin käyttää lääkeaineseulonnassa. Ensimmäisessä osassa tarkoituksena oli kehittää lämpödenaturaatioon perustuva menetelmä, jolla voidaan havaita malliproteiinin stabiiliuuden kasvu, kun ligandi sitoutuu siihen epäspesifisesti. Systeemi kuvaa tilannetta, jossa lääkeaineen sitoutuminen kohdeproteiiniinsa muuttaa proteiinin stabiiliuutta. Toisessa osassa tavoitteena oli kehittää menetelmä, jolla havaitaan peptidi-menetelmällä proteiinien jälkitranslationaalinen modifikaatio

High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target site to block K-Ras driven cancer cell stemness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras stemness-signaling disruptors. A modulated time-resolved Förster resonance energy transfer (mTR-FRET)-assay was developed and benchmarked against an identically designed fluorescence anisotropy (FA)-assay. In both assays, two CaM-binding peptides were labeled with Eu(III)-chelate or fluorescein and used as single-label reporter probes that were displaced from CaM upon competitor binding. Thus, peptidic and small molecule competitors with nanomolar to micromolar affinities to CaM could be detected, including a peptide that was derived from the C-terminus of K-Ras. In order to detect CaM-residue specific covalent inhibitors, a cell lysate-based Förster resonance energy transfer (FRET)-assay was furthermore established. This assay enabled us to measure the slow, residue-specific, covalent inhibition by ophiobolin A in the presence of other endogenous proteins. In conclusion, we have developed a panel of fluorescence-assays that allows identification of conventional and covalent CaM-inhibitors as potential disruptors of K-Ras driven cancer cell stemness

Luminometric Label Array for Counting and Differentiation of Bacteria

Methods for simple and fast detection and differentiation of bacterial species are required, for instance, in medicine, water quality monitoring, and the food industry. Here, we have developed a novel label array method for the counting and differentiation of bacterial species. This method is based on the nonspecific interactions of multiple unstable lanthanide chelates and selected chemicals within the sample leading to a luminescence signal profile that is unique to the bacterial species. It is simple, cost-effective, and/or user-friendly compared to many existing methods, such as plate counts on selective media, automatic (hemocytometer-based) cell counters, flow cytometry, and polymerase chain reaction (PCR)-based methods for identification. The performance of the method was demonstrated with nine single strains of bacteria in pure culture. The limit of detection for counting was below 1000 bacteria per mL, with an average coefficient of variation of 10% achieved with the developed label array. A predictive model was trained with the measured luminescence signals and its ability to differentiate all tested bacterial species from each other, including members of the same genus Bacillus licheniformis and Bacillus subtilis, was confirmed via leave-one-out cross-validation. The suitability of the method for analysis of mixtures of bacterial species was shown with ternary mixtures of Bacillus licheniformis, Escherichia coli JM109, and Lactobacillus reuteri ATCC PTA 4659. The potential future application of the method could be monitoring for contamination in pure cultures; analysis of mixed bacterial cultures, where examining one species in the presence of another could inform industrial microbial processes; and the analysis of bacterial biofilms, where nonspecific methods based on physical and chemical characteristics are required instead of methods specific to individual bacterial species

Molybdenum(VI) complexes with a chiral L-alanine bisphenol [O,N,O,O’] ligand : Synthesis, structure, spectroscopic properties and catalytic activity

Dioxidomolybdenum(VI) compound [MoO2Cl2(dmso)2] reacts with a chiral tetradentate O3N-type L-alanine bisphenol ligand precursor (Et3NH)H2Lala to form an oxidochloridomolybdenum(VI) complex [MoOCl(Lala)] (1) as two separable geometric isomers with phenolate groups in cis or trans positions. The single crystal X-ray and NMR analyses of cis- and trans-1 reveal that the complexes are formed of monomeric molecules, in which the ligand has a tetradentate coordination through three oxygen donors and one nitrogen donor. The reaction of Na2MoO4·2H2O with the same ligand precursor in an acidic methanol solution leads to the formation of an anionic dioxido complex (Et3NH)[MoO2(Lala)] (2) with a trans coordination of the tetradentate ligand. Trans-1 and 2 were studied as active catalysts for olefin epoxidation: i.e. styrene, cyclohexene, S(–)-limonene and (–)-α-pinene using H2O2 and tBuOOH as oxidants.peerReviewe