Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

Ageing and cathepsin inhibition affect the shrinkage of fibre fragments of bovine semitendinosus, biceps femoris and psoas major during heating.

This study examines the effects of ageing (1, 14 days), cathepsin inhibition (No or Yes) and temperature (25-90 °C) on the shrinkage of fibre fragments from three bovine muscles (semitendinosus, biceps femoris and psoas major) during heating. Shrinkage was quantified using light microscopy images. Muscle fibres (except in psoas major) had greater transverse shrinkage, and less longitudinal shrinkage in aged than in unaged muscles at temperatures ≥60-75 °C. In addition, cathepsin inhibition during heating at ≥65-90 °C caused greater transverse shrinkage in semitendinosus fibres, and reduced longitudinal shrinkage for all muscles. At temperatures ≥75 °C, the longitudinal and transverse shrinkage of the fibres was correlated for all muscles. Ageing of biceps femoris increases volume shrinkage on a fibre level, and hence potentially cooking loss, while cathepsin activity in the semitendinosus reduces volume shrinkage. In conclusion, cathepsin activity and ageing influence the shrinkage that occurs during heating and these factors should be explored further to enable optimisation of thermal meat processing.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to ß-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.

Profiling the Nucleobase and Structure Selectivity of Anticancer Drugs and other DNA Alkylating Agents by RNA Sequencing.

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next-generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane.

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2ß1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2ß1 and α1ß1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.

Covalent modifier-type aggregation inhibitor of amyloid-ß based on a cyclo-KLVFF motif.

Inhibition of amyloid-ß (Aß) aggregation could be a drug development target for treating Alzheimer disease. Insufficient activity to inhibit aggregation, however, remains a key issue. Here, we report a covalent modifier-type aggregation inhibitor of Aß, diazirine-equipped cyclo-KLVF(ß-Ph)F (2). Due to the affinity of the cyclo-KLVFF motif for Aß, 2 selectively reacted with Aß1-42 under UV-light irradiation to form an irreversible covalent bond. The Tyr-10 residue of Aß1-42 was identified as the covalent modification site with 2. The extent of cross-ß-sheet structure, characteristics of amyloid aggregation, and toxicity of Aß1-42 were strongly attenuated by this chemical modification.

Photoaffinity labeling the propofol binding site in GLIC.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.

Species selective diazirine positioning in tag-free photoactive quorum sensing probes.

The synthesis and comparison of activities of 'tag-free' probes with diazirines at various positions are described. Remarkable differences in their effects on P. aeruginosa and on human bronchial epithelial cells were observed, supporting the efforts to isolate and identify receptors for N-acyl homoserine lactones.

Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels early oligomer states of the Alzheimer disease Aß42 protein.

The oligomerization of the amyloid-ß protein (Aß) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aß and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aß42 form of the protein. The mass spectrum of a mixture of PADK and Aß42 clearly shows that PADK binds directly to Aß42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aß42 dodecamers, but also removes preformed Aß42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aß42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aß42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.

p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol.

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors. p-4-AziC5-Pro inhibits excitatory current responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native nACh receptor-enriched Torpedo membranes. Thus, p-4-AziC5-Pro is a functional general anesthetic that both modulates and photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol binding sites.

p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels.

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidate's, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidate's novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol.

Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABA(A) receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.

Cathepsin B inhibition improves lung injury associated to D-galactosamine/tumor necrosis factor-alpha-induced liver injury in mice.

The present study was designed to investigate the effects of benzyloxicarbonyl-L-phenylalanyl-alanine-fluoromethylketone (Z-FA.FMK), an inhibitor of cathepsin B on lung injury that occurs concurrently with liver injury induced by D-galactosamine/tumor necrosis factor-alpha (D-GalN/TNF-alpha). Four groups of BALB/c male mice were treated as follows: Group 1--mice receiving intravenous (iv) injections of physiological saline; Group 2--administered with 8 mg/kg Z-FA.FMK by iv injection; Group 3--mice treated with 700 mg/kg D-GalN and 15 microg/kg TNF-alpha by sequential intraperitoneal (ip) injection; Group 4--treated with 700 mg/kg D-GalN and 15 microg/kg TNF-alpha by sequential ip injection 1 h after administration with 8 mg/kg Z-FA.FMK. Mice from Groups 3 and 4 were sacrificed 4 h after D-GalN/TNF-alpha injections. The mice treated with D-GalN/TNF-alpha showed lung damage; increased TNF receptor-associated factor immunoreactivity, lipid peroxidation, protein carbonyl content, and lactate dehydrogenase activity; decreased catalase, superoxide dismutase, and paraoxonase activities. Treatment with Z-FA.FMK resulted in an improvement of these alterations in D-GalN/TNF-alpha-administered mice. The apoptotic index of type-II pneumocytes was the almost same in the four study groups, but pneumocytes labeled with proliferating cell nuclear antigen antibody was more numerous in Group 4 mice. Our results show that D-GalN/TNF-alpha results in lung damage without induction of apoptosis. Treatment with Z-FA.FMK stimulates proliferation of type-II pneumocytes and improves degenerative alterations in injured lung occurred with liver injury induced by D-GalN/TNF-alpha.

Photolabeling of tissue transglutaminase reveals the binding mode of potent cinnamoyl inhibitors.

We have recently developed a new class of cinnamoyl derivatives as potent tissue transglutaminase (TG2) inhibitors. Herein, we report the synthesis of a diazirine derivative of these inhibitors and its application to the photolabeling of its binding site on guinea pig liver transglutaminase. Two novel homology models were generated for this commonly studied TG2, which differ in the conformational state they represent. Tryptic digest and mass spectrometric analysis of the photolabeling experiment showed that only residue Cys230 was labeled, and our homology models were used to visualize these results. This visualization suggested that Cys230 is somewhat more solvent-exposed in the "closed" conformation of TG2, compared to the "open" conformation. Docking experiments suggested binding modes consistent with the labeling pattern that would block access to the tunnel leading to the active site, consistent with the observed mode of inhibition. However, while these modeling simulations favored the closed conformation as the target of our cinnamoyl inhibitors, native PAGE experiments indicated the open conformation of the enzyme in fact predominates in the presence of our photolabeling derivative. These results are important for understanding the binding modes of TG2 inhibitors in general and will be critical for the structure-based design of future inhibitors.

Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

An alcohol binding site on the neural cell adhesion molecule L1.

Prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD) in part by disrupting the neural cell adhesion molecule L1. L1 gene mutations cause neuropathological abnormalities similar to those of FASD. Ethanol and 1-butanol inhibit L1-mediated cell-cell adhesion (L1 adhesion), whereas 1-octanol antagonizes this action. To test the hypothesis that there are alcohol binding sites on L1, we used 3-azibutanol and 3-azioctanol, the photoactivatable analogs of 1-butanol and 1-octanol, to photolabel the purified Ig1-4 domain of human L1 (hL1 Ig1-4). 3-Azibutanol (11 mM), like ethanol, inhibited L1 adhesion in NIH/3T3 cells stably transfected with hL1, whereas subanesthetic concentrations of 3-azioctanol (14 microM) antagonized ethanol inhibition of L1 adhesion. 3-Azibutanol (100-1,000 microM) and 3-azioctanol (10-100 microM) photoincorporated into Tyr-418 on Ig4 and into two adjacent regions in the N terminus, Glu-33 and Glu-24 to Glu-27. A homology model of hL1 Ig1-4 (residues 33-422), based on the structure of the Ig1-4 domains of axonin-1, suggests that Glu-33 and Tyr-418 hydrogen-bond at the interface of Ig1 and Ig4 to stabilize a horseshoe conformation of L1 that favors homophilic binding. Furthermore, this alcohol binding pocket lies within 7 A of Leu-120 and Gly-121, residues in which missense mutations cause neurological disorders similar to FASD. These data suggest that ethanol or selected mutations produce neuropathological abnormalities by disrupting the domain interface between Ig1 and Ig4. Characterization of alcohol agonist and antagonist binding sites on L1 will aid in understanding the molecular basis for FASD and might accelerate the development of ethanol antagonists.

Comparison of cysteine peptidase activities in Trichobilharzia regenti and Schistosoma mansoni cercariae.

Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels.

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

Synthesis and biological evaluation of photoaffinity labeled fusidic acid analogues.

Novel photoaffinity labeled fusidic acid analogues were obtained by a synthetic sequence employing a Wittig reaction between a fusidic acid aldehyde and benzyl bromides in the key step. Three commonly used photoreactive groups, benzophenone, trifluoromethyldiazirine, and aryl azide, were used. The photoaffinity labeled fusidic acid analogues demonstrated a potent antibacterial activity (MIC 0.016-4 microg/mL) and therefore represent a potential tool for the elucidation of the interactions between fusidic acid and its receptor EF-G.

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta.

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.