Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide-gated channels.

BACKGROUND AND PURPOSE: The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. EXPERIMENTAL APPROACH: For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. KEY RESULTS: We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions ("A-2x") of Blastocladiella and Catenaria ("Be," "Ca") CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+ -channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K+ -channel BeCNG1 together with BeCyclOp. CONCLUSION AND IMPLICATIONS: We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria.

Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancer.

Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.

Prey for the Proteasome: Targeted Protein Degradation-A Medicinal Chemist's Perspective.

Targeted protein degradation (TPD), the ability to control a proteins fate by triggering its degradation in a highly selective and effective manner, has created tremendous excitement in chemical biology and drug discovery within the past decades. The TPD field is spearheaded by small molecule induced protein degradation with molecular glues and proteolysis targeting chimeras (PROTACs) paving the way to expand the druggable space and to create a new paradigm in drug discovery. However, besides the therapeutic angle of TPD a plethora of novel techniques to modulate and control protein levels have been developed. This enables chemical biologists to better understand protein function and to discover and verify new therapeutic targets. This Review gives a comprehensive overview of chemical biology techniques inducing TPD. It explains the strengths and weaknesses of these methods in the context of drug discovery and discusses their future potential from a medicinal chemist's perspective.

An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist.

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain.

The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsin-guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.

Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae.

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.

The rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling.

Blastocladiomycota fungi form motile zoospores that are guided by sensory photoreceptors to areas of optimal light conditions. We showed that the microbial rhodopsin of Blastocladiella emersonii is a rhodopsin-guanylyl cyclase (RhGC), a member of a previously uncharacterized rhodopsin class of light-activated enzymes that generate the second messenger cyclic guanosine monophosphate (cGMP). Upon application of a short light flash, recombinant RhGC converted within 8 ms into a signaling state with blue-shifted absorption from which the dark state recovered within 100 ms. When expressed in Xenopus oocytes, Chinese hamster ovary cells, or mammalian neurons, RhGC generated cGMP in response to green light in a light dose-dependent manner on a subsecond time scale. Thus, we propose RhGC as a versatile tool for the optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences.

Upgrading a microplate reader for photobiology and all-optical experiments.

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm(-2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.

Change in protein-ligand specificity through binding pocket grafting.

Recognition and discrimination of small molecules are crucial for biological processes in living systems. Understanding the mechanisms that underlie binding specificity is of particular interest to synthetic biology, e.g. the engineering of biosensors with de novo ligand affinities. Promising scaffolds for such biosensors are the periplasmic binding proteins (PBPs) due to their ligand-mediated structural change that can be translated into a physically measurable signal. In this study we focused on the two homologous polyamine binding proteins PotF and PotD. Despite their structural similarity, PotF and PotD have different binding specificities for the polyamines putrescine and spermidine. To elucidate how specificity is determined, we grafted the binding site of PotD onto PotF. The introduction of 7 mutations in the first shell of the binding pocket leads to a swap in the binding profile as confirmed by isothermal titration calorimetry. Furthermore, the 1.7Å crystal structure of the new variant complexed with spermidine reveals the interactions of the specificity determining residues including a defined water network. Altogether our study shows that specificity is encoded in the first shell residues of the PotF binding pocket and that transplantation of these residues allows the swap of the binding specificity.

Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum.

Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.

Structure and glycolipid binding properties of the nematicidal protein Cry5B.

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g., hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids) and also suggests that domain II is important for receptor binding. We found that not only galactose but also N-acetylgalactosamine (GalNAc) is an efficient competitor for binding between Cry5B and glycolipids. GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with a specificity equal to that of activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.