Protein Of Interest Research Articles

Bifunctional Peptide Nanofibrils for Targeted Protein Degradation.

Proteolysis targeting chimera (PROTAC) is a state-of-the-art technology for ablating undruggable targets. A PROTAC degrader achieves targeted protein degradation (TPD) through the simultaneous binding of a protein of interest (POI) and an E3 ligase to form a ternary complex. A nanofibril-based PROTAC strategy to form a polynary (E3)m : PROTAC : (POI)n complex has not been reported in the TPD field up to this point. A recent innovation shows that a POI ligand and E3 ligase ligand don't have to be within a fused degrader molecule. Instead, they can be recruited to cellular proximity by a self-assembly-driving peptide and click chemistry. The resulting nanofibrils can recruit multiple POI and E3 ligase molecules to form a polynary complex as a degradation center. The so-called Nano-PROTAC provides a novel approach for TPD in cancer therapy.

Bifunctional Peptide Nanofibrils for Targeted Protein Degradation

AbstractProteolysis targeting chimera (PROTAC) is a state‐of‐the‐art technology for ablating undruggable targets. A PROTAC degrader achieves targeted protein degradation (TPD) through the simultaneous binding of a protein of interest (POI) and an E3 ligase to form a ternary complex. A nanofibril‐based PROTAC strategy to form a polynary (E3)m : PROTAC : (POI)n complex has not been reported in the TPD field up to this point. A recent innovation shows that a POI ligand and E3 ligase ligand don't have to be within a fused degrader molecule. Instead, they can be recruited to cellular proximity by a self‐assembly‐driving peptide and click chemistry. The resulting nanofibrils can recruit multiple POI and E3 ligase molecules to form a polynary complex as a degradation center. The so‐called Nano‐PROTAC provides a novel approach for TPD in cancer therapy.

Abstract A055: Discovery of a novel, highly potent VHL-recruiting EZH2 PROTAC degrader targeting MLL-r AML

Abstract Mutated or overexpressed Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), has been implicated in numerous cancers, including breast, leukemia, and lung. Through its methyltransferase activity, EZH2 tri-methylates H3K27 to repress transcription. In addition to its enzymatic activity, recent studies have highlighted non-enzymatic oncogenic functions of EZH2 that limit the efficacy of enzymatic inhibitors. Therefore, alternative therapeutic strategies such as proteolysis targeting chimera (PROTAC) degraders that suppress both enzymatic and non-enzymatic activity have been developed. PROTACs are heterobifunctional molecules consisting of a protein of interest (POI) binder linked to an E3-ligase ligand. The manufactured proximity between the POI and the recruited E3-ligase complex initiates ubiquitination of the POI, resulting in its degradation via the 26S proteasome. EZH2 exhibits enzymatic and non-enzymatic oncogenic activity in various cancers including MLL1-rearranged acute myeloid leukemia (MLL-r AML) and triple negative breast cancer (TNBC). Here we present MS8847, a novel VHL-recruiting EZH2 PROTAC degrader. In MLL-r AML cells, MS8847 induces potent EZH2 degradation in a ubiquitin proteasome-, time- and concentration-dependent manner. Compared to the previously published EZH2-targeting PROTAC degraders (MS8815, YM281, U3i, and E7) and parent inhibitor, Tazemetostat, MS8847 displays superior EZH2 degradation and cell growth inhibition in MLL-r AML cell lines dependent on both enzymatic and non-enzymatic EZH2 activity. MS8847 also induces EZH2 degradation and cell growth inhibition in TNBC cell lines. Moreover, MS8847 has a favorable PK profile making it suitable for in vivo studies. Together, MS8847 offers an improved VHL-recruiting EZH2 PROTAC degrader with potential as an effective therapeutic agent to treat EZH2-dependent cancers. Citation Format: Julia Velez, Xufen Yu, Brandon Dale, Kwang-Su Park, H. Ümit Kaniskan, Jian Jin. Discovery of a novel, highly potent VHL-recruiting EZH2 PROTAC degrader targeting MLL-r AML [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A055.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.

Discovery of proteolysis-targeting chimera targeting undruggable proteins using a covalent ligand screening approach

Targeted protein degradation (TPD) technology, such as proteolysis-targeting chimera (PROTAC), has become a new therapeutic modality. However, the degradation of undruggable proteins, such as those involved in protein-protein interactions (PPIs), using PROTAC is still limited owing to the difficulties in finding small-molecule binders of these proteins. To identify new chemical moieties that bind to the target sites of the protein of interest (POI), we conducted a site-specific and fragment-based covalent ligand screening using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To apply the selected hits to the PROTAC approach, two-dimensional (2D) nuclear magnetic resonance (NMR) experiments were performed to evaluate the reversible binding of their analogs without covalent warheads. To proof the proposed approach, human mouse double minute (MDM)2 was selected as a model system since it is involved in PPIs and is known to be a degradable target protein. Western blot analysis showed that newly synthesized PROTACs, incorporated reversible analogs of screening hits, affected degradation in a dose- and time-dependent manner. This methodology makes it possible to use PROTAC technology to exploit previously undruggable proteins for TPD.

Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging.

This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed in vitro, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for in vitro and in vivo imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or in vivo biological activities of the POI.

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification.

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.

Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

AbstractThe emergence of thiol‐mediated uptake (TMU) as a powerful strategy to penetrate cells calls for the development of practical small‐molecule TMU tools for traceless delivery. Toward this goal, esters are explored here as bioreversible linkers between dynamic covalent cascade exchangers accounting for TMU and the protein of interest (POI). The method relies on α‐aryl‐α‐diazoamides that react with carboxylic acids of the POI to form esters that can be enzymatically hydrolyzed inside cells to release the native POI. A two‐step protocol is established for bioreversible conjugation of TMU tags to the POI. Despite the small number of tags attached to POIs to prevent isoelectric precipitation, POIs with traceless TMU tags are shown to efficiently enter cells not only in 2D culture but also in 3D spheroids mimicking deep tissue, confirming a key advantage of TMU. Uptake inhibition by various thiol‐reactive agents confirms the participation of cell‐surface thiols in cell penetration, i. e., the occurrence of TMU.

Bioorthogonal Aptamer-ATTEC Conjugates for Degradation of Alpha-Synuclein via Autophagy-Lysosomal Pathway.

Autophagosome-tetheringcompound (ATTEC) technology has recently been emerging as a novel approach for degrading proteins of interest (POIs). However, it still faces great challenges in how to design target-specific ATTEC molecules. Aptamers are single-stranded DNA or RNA oligonucleotides that can recognize their target proteins with high specificity and affinity. Here, ATTEC is combined with aptamers for POIs degradation. As a proof of concept, pathological protein α-synuclein (α-syn) is chosen as the target and an efficient α-syn degrader is generated. Aptamer as a targeting warhead of α-syn is conjugated with LC3B-binding compound 5,7-dihydroxy-4-phenylcoumarin (DP) via bioorthogonal click reaction. It is demonstrated that the aptamer conjugated with DP is capable of clearing α-syn through LC3 and autophagic degradation. These results indicate that aptamer-based ATTECs are a versatile approach to degrade POIs by taking advantage of the well-defined different aptamers for targeting diverse proteins, which provides a new way for the design of ATTECs to degradation of targeted proteins.

B-291 Proximity Labeling at Sites of Protein-Protein Interactions Using a Switchable DNA Catalyst

Abstract Background Extracellular protein-protein interactions (PPIs) play a crucial role in cell-cell adhesion, communication, and signaling. The dysregulation of PPIs in cancer caused by differential protein expression can drive oncogenic factors. Traditional methods to study PPIs disfavor weak or transient interacting protein partners, leaving them undiscovered. Proximity labeling (PL) is an emerging tool in which a catalyst is attached to a protein of interest (POI) and creates reactive species that tag nearby endogenous proteins. Tagged proteins are subsequently isolated and identified as potential interacting partners with the POI. If PL could be activated at a more specific subcellular location, such as the site of a PPI, more detailed information could be unveiled concerning the interactome of cancer-relevant PPIs. Recent advances in PL involve the use of a synthetic catalyst that can be localized to a POI via an antibody or ligand and avoids the need for PL enzyme-POI fusion proteins to be constructed, allowing for analysis of primary tissue samples. Methods To create a PL catalyst with pseudo-allosteric regulation, we created a switchable DNA catalyst. The DNA catalyst is composed of synthetic photocatalyst and quencher moieties covalently linked to DNA oligomers such that when hybridized together the photocatalyst is quenched and inactive. The DNA scaffold affords conformational control that is utilized to change the distance between photocatalyst and quencher, thus regulating photocatalyst activity. Split triggers composed of DNA oligos that bind to catalyst-DNA and simultaneously disrupt quencher-DNA hybridization were used to create an AND logic gate where both split triggers need to be in proximity to activate the PL catalyst. The split triggers were localized to POIs via connection to aptamers, short pieces of synthetic DNA that bind targets. Using the c-Met homodimer as a model system, we first labeled c-Met expressing HEK293T cells with aptamer-split trigger DNA oligomers as well as the quenched photocatalyst. After the catalyst was activated at the site of the PPI, we performed proximity labeling using a biotin-phenol substrate in the presence of an oxidizing agent and 450 nm light. Fluorescent streptavidin was used to visualize sites of biotinylation and detected using fluorescence microscopy. Results We observed extracellular biotin signal only of c-Met transfected cells as evidenced by the expression of a co-transfection plasmid encoding a fluorescent nuclear protein. When one of the split triggers had the aptamer motif removed, meaning it couldn’t be localized to the cell surface, no labeling was observed which shows that both split probes must be localized to the cell surface to activate labeling. Additionally, controls including omission of light, photocatalyst, or biotin phenol showed no labeling. Using a free-floating version of the photocatalyst to label cells, we confirmed that lysates contained biotinylated proteins via western blot. Conclusion We localized two split triggers to interacting proteins to activate a PL catalyst and performed proximity labeling at the site of the PPI. This technique demonstrates non-genetic split proximity labeling to enable downstream analysis of cancer cell proteomes in an ultra-specific manner and expands the toolkit for spatiotemporally controlled proximity labeling.

Selective Protein of Interest Degradation through the Split-and-Mix Liposome Proteolysis Targeting Chimera Approach.

Proteolysis Targeting Chimera (PROTAC) technology represents a promising new approach for target protein degradation using a cellular ubiquitin-proteasome system. Recently, we developed a split-and-mix nanoplatform based on peptide self-assembly, which could serve as a self-adjustable platform for multifunctional applications. However, the lower drug efficacy limits further biomedical applications of peptide-based SM-PROTAC. In this study, we develop a novel split-and-mix PROTAC system based on liposome self-assembly (LipoSM-PROTAC), concurrent with modification of FA (folate) to enhance its tumor-targeting capabilities. Estrogen receptors (ERα) were chosen as the protein of interest (POI) to validate the efficacy of Lipo degraders. Results demonstrate that this PROTAC can be efficiently and selectively taken up into the cells by FA receptor-positive cells (FR+) and degrade the POI with significantly reduced concentration. Compared to the peptide-based SM-PROTACs, our designed LipoSM-PROTAC system could achieve therapeutic efficacy with a lower concentration and provide opportunities for clinical translational potential. Overall, the LipoSM-based platform shows a higher drug efficacy, which offers promising potential applications for PROTAC and other biomolecule regulations.

3D based generative PROTAC linker design with reinforcement learning.

Proteolysis targeting chimera (PROTAC), has emerged as an effective modality to selectively degrade disease-related proteins by harnessing the ubiquitin-proteasome system. Due to PROTACs' hetero-bifunctional characteristics, in which a linker joins a warhead binding to a protein of interest (POI), conferring specificity and a E3-ligand binding to an E3 ubiquitin ligase, this could trigger the ubiquitination and transportation of POI to the proteasome, followed by degradation. The rational PROTAC linker design is challenging due to its relatively large molecular weight and the complexity of maintaining the binding mode of warhead and E3-ligand in the binding pockets of counterpart. Conventional linker generation method can only generate linkers in either 1D SMILES or 2D graph, without taking into account the information of ternary structures. Here we propose a novel 3D linker generative model PROTAC-INVENT which can not only generate SMILES of PROTAC but also its 3D putative binding conformation coupled with the target protein and the E3 ligase. The model is trained jointly with the RL approach to bias the generation of PROTAC structures toward pre-defined 2D and 3D based properties. Examples were provided to demonstrate the utility of the model for generating reasonable 3D conformation of PROTACs. On the other hand, our results show that the associated workflow for 3D PROTAC conformation generation can also be used as an efficient docking protocol for PROTACs.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

Chalcone derivatives as dual-sensitive fluorescent probe for protein aggregation investigation

Protein homeostasis is of great importance for cellular viability. However, environmental factors such as drug treatment, pathogen infection etc. may cause compromised proteostasis, which further lead to protein misfolding or aggregation. Herein, based on rational design, a toolbox of chalcone derivatives was developed to probe the whole process of protein aggregation. The chalcone derivatives disclosed here were dual-sensitive probes that exhibited viscosity-dependent fluorescence intensity enhancement and polarity-dependent emission wavelength variability. Structural modulation identified that both polarity and viscosity sensitivity of chalcone derivatives were tunable via electron density regulation and π-rich bridge extension way. Furthermore, upon binding to protein of interest (POI), the optimized probe demonstrated “stepwise turn-on” fluorescence performances and varying wavelength in different aggregating stages that allowed for the investigation of microenvironment change of the whole process of protein aggregation. Finally, the optimized probe was utilized to investigate the aggregated proteome in live cells upon proteasome inhibitor stressing. Overall, a rational approach was disclosed here to develop new probes with controllable polarity and viscosity sensitivity, tunable spectral coverage for efficient investigation of protein aggregation in live cell.

Targeted Protein Degradation: Advances, Challenges, and Prospects for Computational Methods.

The therapeutic approach of targeted protein degradation (TPD) is gaining momentum due to its potentially superior effects compared with protein inhibition. Recent advancements in the biotech and pharmaceutical sectors have led to the development of compounds that are currently in human trials, with some showing promising clinical results. However, the use of computational tools in TPD is still limited, as it has distinct characteristics compared with traditional computational drug design methods. TPD involves creating a ternary structure (protein-degrader-ligase) responsible for the biological function, such as ubiquitination and subsequent proteasomal degradation, which depends on the spatial orientation of the protein of interest (POI) relative to E2-loaded ubiquitin. Modeling this structure necessitates a unique blend of tools initially developed for small molecules (e.g., docking) and biologics (e.g., protein-protein interaction modeling). Additionally, degrader molecules, particularly heterobifunctional degraders, are generally larger than conventional small molecule drugs, leading to challenges in determining drug-like properties like solubility and permeability. Furthermore, the catalytic nature of TPD makes occupancy-based modeling insufficient. TPD consists of multiple interconnected yet distinct steps, such as POI binding, E3 ligase binding, ternary structure interactions, ubiquitination, and degradation, along with traditional small molecule properties. A comprehensive set of tools is needed to address the dynamic nature of the induced proximity ternary complex and its implications for ubiquitination. In this Perspective, we discuss the current state of computational tools for TPD. We start by describing the series of steps involved in the degradation process and the experimental methods used to characterize them. Then, we delve into a detailed analysis of the computational tools employed in TPD. We also present an integrative approach that has proven successful for degrader design and its impact on project decisions. Finally, we examine the future prospects of computational methods in TPD and the areas with the greatest potential for impact.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti-Hook Effect for Tumor Therapy.

Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target induced unintended tissue effects and intrinsic "hook effect" hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano-PROTACs) modality with a center-spoke degradation network for achieving efficient dose-dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self-assemble into Nano-PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano-PROTACs can form effective polynary complexes and E3 ligases degradation network with multi-binding sites, achieving dose-dependent protein degradation with "anti-hook effect". The generality and efficacy of Nano-PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide-range dose-dependent manner with a 95 % degradation rate and long-lasting potency up to 72 h in vitro. Significantly, Nano-PROTACs achieve in vivo dose-dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self-assembly strategy, the Nano-PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti‐Hook Effect for Tumor Therapy

AbstractProteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post‐translational protein degradation capabilities. However, off‐target induced unintended tissue effects and intrinsic “hook effect” hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano‐PROTACs) modality with a center‐spoke degradation network for achieving efficient dose‐dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self‐assemble into Nano‐PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano‐PROTACs can form effective polynary complexes and E3 ligases degradation network with multi‐binding sites, achieving dose‐dependent protein degradation with “anti‐hook effect”. The generality and efficacy of Nano‐PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide‐range dose‐dependent manner with a 95 % degradation rate and long‐lasting potency up to 72 h in vitro. Significantly, Nano‐PROTACs achieve in vivo dose‐dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self‐assembly strategy, the Nano‐PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Hydrophobic Tag Tethering Degradation, The Emerging Targeted Protein Degradation Strategy.

Targeted protein degradation (TPD) strategies have become a new trend in drug discovery due to the capability of triggering the degradation of protein of interest (POI) selectively and effectively in recent decades. Particularly, the hydrophobic tag tethering degrader (HyTTD) has drawn a lot of attention and may offer a promising strategy for new drug research and development in the future. Herein, we will give an overview of the development of HyTTD, the structure-activity relationship (SAR) between HyTTD and linkers, HyTs, and ligand motifs, as well as the various HyTTDs targeting different targets, thus offering a rational strategy for the design of HyTTDs in further TPD drug discovery.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells

BackgroundA commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters.ResultsAs a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable.ConclusionsWe conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.