Targeted genetic and small molecule disruption of N-Ras CaaX cleavage alters its localization and oncogenic potential.

Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.

Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.

Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.

Discovery of 2-thiobenzimidazoles as noncovalent inhibitors of SARS-CoV-2 main protease.

The discovery of antiviral agents against SARS-CoV-2 is an important step toward ending the COVID-19 pandemic and to tackle future outbreaks. In this context, the main protease (Mpro) represents an ideal target for developing coronavirus antivirals, being conserved among different strains and essential for survival. In this work, using in silico tools, we created and validated a docking protocol able to predict binders to the catalytic site of Mpro. The following structure-based virtual screening of a subset of the ZINC library (over 4.3 million unique structures), led to the identification of a hit compound having a 2-thiobenzimidazole scaffold. The inhibitory activity was confirmed using a FRET-based proteolytic assay against recombinant Mpro. Structure-activity relationships were obtained with the synthesis of a small library of analogs, guided by the analysis of the docking pose. Our efforts led to the identification of a micromolar Mpro inhibitor (IC50 = 14.9 µM) with an original scaffold possessing ideal drug-like properties (predicted using the QikProp function) and representing a promising lead for the development of a novel class of coronavirus antivirals.

Signaling-Biased and Constitutively Active Dopamine D2 Receptor Variant.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1ß1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a ∼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.

Measuring Cellular Ion Transport by Magnetoencephalography.

The cellular-level process of ion transport is known to generate a magnetic field. A noninvasive magnetoencephalography (MEG) technique was used to measure the magnetic field emanating from HeLa, HEK293, and H9c2(2-1) rat cardiac cells. The addition of a nonlethal dose of ionomycin to HeLa and capsaicin to TRPV1-expressing HEK293 cells resulted in a sudden change in the magnetic field signal consistent with Ca2+ influx, which was also observed by confocal fluorescence microscopy under the same conditions. In contrast, addition of capsaicin to TRPV1-expressing HEK293 cells containing an optimum amount of a TRPV1 antagonist (ruthenium red), resulted in no detectable magnetic or fluorescent signals. These signals confirmed that the measured MEG signals are due to cellular ion transport through the cell membrane. In general, there is evidence that ion channel/transporter activation and ionic flux are linked to cancer. Therefore, our work suggests that MEG could represent a noninvasive method for detecting cancer.

Photoactivatable Dopamine and Sulpiride to Explore the Function of Dopaminergic Neurons and Circuits.

Kinetic analysis of dopamine receptor activation and inactivation and the study of dopamine-dependent signaling requires precise simulation of the presynaptic release of the neurotransmitter dopamine and tight temporal control over the release of dopamine receptor antagonists. The 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting group was conjugated to dopamine and the dopamine receptor antagonist sulpiride to generate "caged" versions of these neuromodulators (CyHQ-O-DA and CyHQ-sulpiride, respectively) that could release their payloads with 365 or 405 nm light or through 2-photon excitation (2PE) at 740 nm. These compounds are stable under physiological conditions in the dark, yet photolyze rapidly and cleanly to yield dopamine or sulpiride and the caging remnant CyHQ-OH. CyHQ-O-DA mediated the light activation of dopamine-1 (D1) receptors on the breast cancer cell line MDA-MB-231 in culture. In mouse brain slice from the substantia nigra pars compacta, localized flash photolysis of CyHQ-O-DA accurately mimicked the natural presynaptic release of dopamine and activation of dopamine-2 (D2) receptors, causing a robust, concentration-dependent, and repeatable G protein-coupled inwardly rectifying potassium channel-mediated outward current in whole-cell voltage clamp recordings that was amplified by cocaine and blocked by sulpiride. Photolysis of CyHQ-sulpiride rapidly blocked synaptic activity, enabling measurement of the unbinding rates of dopamine and quinpirole, a D2 receptor agonist. These tools will enable more detailed study of dopamine receptors, their interactions with other GPCRs, and the physiology of dopamine signaling in the brain.

Rce1: mechanism and inhibition.

Ras converting enzyme 1 (Rce1) is an integral membrane endoprotease localized to the endoplasmic reticulum that mediates the cleavage of the carboxyl-terminal three amino acids from CaaX proteins, whose members play important roles in cell signaling processes. Examples include the Ras family of small GTPases, the γ-subunit of heterotrimeric GTPases, nuclear lamins, and protein kinases and phosphatases. CaaX proteins, especially Ras, have been implicated in cancer, and understanding the post-translational modifications of CaaX proteins would provide insight into their biological function and regulation. Many proteolytic mechanisms have been proposed for Rce1, but sequence alignment, mutational studies, topology, and recent crystallographic data point to a novel mechanism involving a glutamate-activated water and an oxyanion hole. Studies using in vivo and in vitro reporters of Rce1 activity have revealed that the enzyme cleaves only prenylated substrates and the identity of the a2 amino residue in the Ca1a2X sequence is most critical for recognition, preferring Ile, Leu, or Val. Substrate mimetics can be somewhat effective inhibitors of Rce1 in vitro. Small-molecule inhibitor discovery is currently limited by the lack of structural information on a eukaryotic enzyme, but a set of 8-hydroxyquinoline derivatives has demonstrated an ability to mislocalize all three mammalian Ras isoforms, giving optimism that potent, selective inhibitors might be developed. Much remains to be discovered regarding cleavage specificity, the impact of chemical inhibition, and the potential of Rce1 as a therapeutic target, not only for cancer, but also for other diseases.

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells.

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.

Photoactivatable, biologically-relevant phenols with sensitivity toward 2-photon excitation.

Spatio-temporal release of biologically relevant small molecules provides exquisite control over the activation of receptors and signaling pathways. This can be accomplished via a photochemical reaction that releases the desired small molecule in response to irradiation with light. A series of biologically-relevant signaling molecules (serotonin, octopamine, capsaicin, N-vanillyl-nonanoylamide, estradiol, and tyrosine) that contain a phenol moiety were conjugated to the 8-bromo-7-hydroxyquinolinyl (BHQ) or 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting groups (PPGs). The CyHQ caged compounds proved sensitive toward 1PE and 2PE processes with quantum efficiencies of 0.2-0.4 upon irradiation at 365 nm and two-photon action cross sections of 0.15-0.31 GM when irradiated at 740 nm. All but one BHQ caged compound, BHQ-estradiol, were found to be sensitive to photolysis through 1PE and 2PE with quantum efficiencies of 0.30-0.40 and two photon cross sections of 0.40-0.60 GM. Instead of releasing estradiol, BHQ-estradiol underwent debromination.

Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases.

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A 'warhead' free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.

Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones.

The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.

Inhibition of kinesin motor proteins by adociasulfate-2.

Kinesin motor proteins are involved in cell division and intracellular transport of vesicles and organelles, and as such, they play a role in neurological disease, cancer, and developmental disorders. Inhibitors of kinesin would be valuable as probes of cell physiology and as potential therapeutics. Adociasulfate-2 (AS-2) is the only known natural product inhibitor of kinesins, but its mechanism of action is unknown. We utilized kinetic studies, dynamic light scattering, and transmission electron microscopy to investigate the inhibitory action of AS-2. Our data suggest that AS-2 is not a classical 1:1 inhibitor. Instead, a rodlike aggregate that mimics microtubules is complexed with kinesin and inhibits its ATPase activity. An intriguing implication of this hypothesis is that aggregates of a chiral natural product can have interesting and biologically relevant properties. This mode of action might represent one way in which a small molecule can disrupt a protein-protein interaction.

Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.

Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H(2)O(2)-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.