E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity.

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

Fragment-Based Ligand Discovery Using Protein-Observed 19F NMR: A Second Semester Organic Chemistry CURE Project.

Curriculum-based undergraduate research experiences (CUREs) have been shown to increase student retention in STEM fields and are starting to become more widely adopted in chemistry curricula. Here we describe a 10-week CURE that is suitable for a second-semester organic chemistry laboratory course. Students synthesize small molecules and use protein-observed 19F (PrOF) NMR to assess the small molecule's binding affinity to a target protein. The research project introduced students to multistep organic synthesis, structure-activity relationship studies, quantitative biophysical measurements (measuring Kd from PrOF NMR experiments), and scientific literacy. Docking experiments could be added to help students understand how changes in a ligand structure may affect binding to a protein. Assessment using the CURE survey indicates self-perceived skill gains from the course that exceed gains measured in a traditional and an inquiry-based laboratory experience. Given the speed of the binding experiment and the alignment of the synthetic methods with a second-semester organic chemistry laboratory course, a PrOF NMR fragment-based ligand discovery lab can be readily implemented in the undergraduate chemistry curriculum.

Dual Labeling of the CBP/p300 KIX Domain for 19 F†NMR Leads to Identification of a New Small-Molecule Binding Site.

Protein-Observed Fluorine NMR (PrOF NMR) spectroscopy is an emerging technique for screening and characterizing small-molecule-protein interactions. The choice of which amino acid to label for PrOF NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small-molecule compounds elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1 H,15 N HSQC/HMQC NMR spectroscopy. Metabolic labeling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription-factor protein interactions with small-molecule ligands.

Quantifying Protein Concentrations Using Smartphone Colorimetry: A New Method for an Established Test.

Proteins are involved in nearly every biological process, which makes them of interest to a range of scientists. Previous work has shown that hand-held cameras can be used to determine the concentration of colored analytes in solution, and this paper extends the approach to reactions involving a color change in order to quantify protein concentration (e.g., green to blue). Herein, we describe the successful use of smartphone colorimetry to quantify protein concentration using two common colorimetric biochemical methods, the Bradford and biuret assays. The ease of the experimental setup makes these lab experiments accessible to a wide range of students and can be used as both high school and college level laboratory experiments.

Fragment screening and druggability assessment for the CBP/p300 KIX domain through protein-observed 19F†NMR spectroscopy.

(19)F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein-protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed (19)F NMR spectroscopy. Screening of 508 compounds and validation by (1)H-(15)N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or "druggability" of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.

Fragment-Based NMR Screening of the BPTF PHD Finger Methyl Lysine Reader Leads to the First Small-Molecule Inhibitors.

Methyl lysine readers, specifically PHD fingers, are emerging epigenetic targets in human diseases. For example, several PHD finger fusions are implicated in clinical cases of acute myeloid leukemia, highlighting the potential for PHD inhibitors in disease regulation. However, limited chemical matter targeting PHD fingers exists. Here we report the first fragment-based screen against the BPTF PHD to identify several of the first reported BPTF PHD-targeting small-molecule ligands. We used ligand-observed NMR to first screen a fragment library, followed by biophysical validation to prioritize two scaffolds, pyrrolidine- and pyridazine-containing fragments. Structural predictions show that these respective scaffolds may engage two distinct subpockets on the protein. The demonstrated ligandability of the BPTF PHD supports the future development of methyl lysine reader chemical probes to study their oncogenic functions.

Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket.

The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

A Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes.

Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds 26 and 30 have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.

Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases.

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.

Discovery and Characterization of the Antimetabolite Action of Thioacetamide-Linked 1,2,3-Triazoles as Disruptors of Cysteine Biosynthesis in Gram-Negative Bacteria.

Increasing rates of drug-resistant Gram-negative (GN) infections, combined with a lack of new GN-effective antibiotic classes, are driving the need for the discovery of new agents. Bacterial metabolism represents an underutilized mechanism of action in current antimicrobial therapies. Therefore, we sought to identify novel antimetabolites that disrupt key metabolic pathways and explore the specific impacts of these agents on bacterial metabolism. This study describes the successful application of this approach to discover a new series of chemical probes, N-(phenyl)thioacetamide-linked 1,2,3-triazoles (TAT), that target cysteine synthase A (CysK), an enzyme unique to bacteria that is positioned at a key juncture between several fundamental pathways. The TAT class was identified using a high-throughput screen against Escherichia coli designed to identify modulators of pathways related to folate biosynthesis. TAT analog synthesis demonstrated a clear structure-activity relationship, and activity was confirmed against GN antifolate-resistant clinical isolates. Spontaneous TAT resistance mutations were tracked to CysK, and mode of action studies led to the identification of a false product formation mechanism between the CysK substrate O-acetyl-l-serine and the TATs. Global transcriptional responses to TAT treatment revealed that these antimetabolites impose substantial disruption of key metabolic networks beyond cysteine biosynthesis. This study highlights the potential of antimetabolite drug discovery as a promising approach to the discovery of novel GN antibiotics and the pharmacological promise of TAT CysK probes.

Oxygen Sensing with Perfluorocarbon-Loaded Ultraporous Mesostructured Silica Nanoparticles.

Oxygen homeostasis is important in the regulation of biological function. Disease progression can be monitored by measuring oxygen levels, thus producing information for the design of therapeutic treatments. Noninvasive measurements of tissue oxygenation require the development of tools with minimal adverse effects and facile detection of features of interest. Fluorine magnetic resonance imaging (19F MRI) exploits the intrinsic properties of perfluorocarbon (PFC) liquids for anatomical imaging, cell tracking, and oxygen sensing. However, the highly hydrophobic and lipophobic properties of perfluorocarbons require the formation of emulsions for biological studies, though stabilizing these emulsions has been challenging. To enhance the stability and biological loading of perfluorocarbons, one option is to incorporate perfluorocarbon liquids into the internal space of biocompatible mesoporous silica nanoparticles. Here, we developed perfluorocarbon-loaded ultraporous mesostructured silica nanoparticles (PERFUMNs) as 19F MRI detectable oxygen-sensing probes. Ultraporous mesostructured silica nanoparticles (UMNs) have large internal cavities (average = 1.8 cm3 g-1), facilitating an average 17% loading efficiency of PFCs, meeting the threshold fluorine concentrations needed for imaging studies. Perfluoro-15-crown-5-ether PERFUMNs have the highest equivalent nuclei per PFC molecule and a spin-lattice (T1) relaxation-based oxygen sensitivity of 0.0032 mmHg-1 s-1 at 16.4 T. The option of loading PFCs after synthesizing UMNs, rather than traditional in situ core-shell syntheses, allows for use of a broad range of PFC liquids from a single material. The biocompatible and tunable chemistry of UMNs combined with the intrinsic properties of PFCs makes PERFUMNs a MRI sensor with potential for anatomical imaging, cell tracking, and metabolic spectroscopy with improved stability.

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen.

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

Paramagnetic relaxation enhancement for protein-observed 19F NMR as an enabling approach for efficient fragment screening.

Protein-observed 19F (PrOF) NMR is an emerging tool for ligand discovery. To optimize the efficiency of PrOF NMR experiments, paramagnetic relaxation enhancement through the addition of chelated Ni(II) was used to shorten longitudinal relaxation time without causing significant line broadening. Thus enhancing relaxation time leads to shorter experiments without perturbing the binding of low- or high-affinity ligands. This method allows for time-efficient screening of potential ligands for a wide variety of proteins in the growing field of fragment-based ligand discovery.

Protein-observed (19)F-NMR for fragment screening, affinity quantification and druggability assessment.

NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed (19)F-NMR (PrOF NMR) using (19)F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.