Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins.

Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2'-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145. For the RNA methyltransferase, the catalytic lysine K6968 that serves as a Brønsted base has significant population of its deprotonated state via strong coupling with K6844 and Y6845. For the main protease, Partial Order Optimum Likelihood (POOL) predicts two clusters of biochemically active residues; one includes the catalytic H41 and C145 and neighboring residues. The other surrounds a second pocket adjacent to the catalytic site and includes S1 residues F140, L141, H163, E166, and H172 and also S2 residue D187. This secondary recognition site could serve as an alternative target for the design of molecular probes. From in silico screening of library compounds, ligands with predicted affinity for the secondary site are reported. For the NSP16-NSP10 complex that comprises the RNA methyltransferase, three different sites are predicted. One is the catalytic core at the conserved K-D-K-E motif that includes catalytic residues D6928, K6968, and E7001 plus K6844. The second site surrounds the catalytic core and consists of Y6845, C6849, I6866, H6867, F6868, V6894, D6895, D6897, I6926, S6927, Y6930, and K6935. The third is located at the heterodimer interface. Ligands predicted to have high affinity for the first or second sites are reported. Three sites are also predicted for the nucleocapsid protein. This work uncovers key interactions that contribute to the function of the three viral proteins and also suggests alternative sites for ligand design.

Design, Synthesis, and Characterization of [18F]mG2P026 as a High-Contrast PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

An array of triazolopyridines based on JNJ-46356479 (6) were synthesized as potential positron emission tomography radiotracers for metabotropic glutamate receptor 2 (mGluR2). The selected candidates 8-10 featured enhanced positive allosteric modulator (PAM) activity (20-fold max.) and mGluR2 agonist activity (25-fold max.) compared to compound 6 in the cAMP GloSensor assays. Radiolabeling of compounds 8 and 9 (mG2P026) was achieved via Cu-mediated radiofluorination with satisfactory radiochemical yield, >5% (non-decay-corrected); high molar activity, >180 GBq/µmol; and excellent radiochemical purity, >98%. Preliminary characterization of [18F]8 and [18F]9 in rats confirmed their excellent brain permeability and binding kinetics. Further evaluation of [18F]9 in a non-human primate confirmed its superior brain heterogeneity in mapping mGluR2 and higher affinity than [18F]6. Pretreatment with different classes of PAMs in rats and a primate led to similarly enhanced brain uptake of [18F]9. As a selective ligand, [18F]9 has the potential to be developed for translational studies.

Electrostatic fingerprints of catalytically active amino acids in enzymes.

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major enzyme commission classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. The catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pKa differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pKa of the acidic residue is higher than that of the histidine. All catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pKa of the anion-forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pKa differences within 1 pH unit. Some evidence of the possible types of interactions that facilitate nucleophilicity is discussed. The interactions reported here provide important clues about how side chain functional groups that are weak Brønsted acids or bases for the free amino acid in solution can achieve catalytic potency and become strong acids, bases or nucleophiles in the enzymatic environment.

Synthesis and Characterization of 5-(2-Fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide as a PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for several neuropsychiatric disorders. An mGluR2 function in etiology could be unveiled by positron emission tomography (PET). In this regard, 5-(2-fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide ([11C]13, [11C]mG2N001), a potent negative allosteric modulator (NAM), was developed to support this endeavor. [11C]13 was synthesized via the O-[11C]methylation of phenol 24 with a high molar activity of 212 ± 76 GBq/µmol (n = 5) and excellent radiochemical purity (>99%). PET imaging of [11C]13 in rats demonstrated its superior brain heterogeneity and reduced accumulation with pretreatment of mGluR2 NAMs, VU6001966 (9) and MNI-137 (26), the extent of which revealed a time-dependent drug effect of the blocking agents. In a nonhuman primate, [11C]13 selectively accumulated in mGluR2-rich regions and resulted in high-contrast brain images. Therefore, [11C]13 is a potential candidate for translational PET imaging of the mGluR2 function.

Molecular structure and spectroscopic characterization of Carbamazepine with experimental techniques and DFT quantum chemical calculations.

A systematic vibrational spectroscopic assignment and analysis of Carbamazepine has been carried out by using FT-IR, FT-Raman and UV spectral data. The vibrational analysis were aided by electronic structure calculations - ab initio (RHF) and hybrid density functional methods (B3LYP) performed with standard basis set 6-31G(d,p). Molecular equilibrium geometries, electronic energies, natural bond order analysis, harmonic vibrational frequencies and IR intensities have been computed. A detailed interpretation of the vibrational spectra of the molecule has been made on the basis of the calculated Potential Energy Distribution (PED) by VEDA program. UV-visible spectrum of the compound was also recorded and the electronic properties, such as HOMO and LUMO energies and λmax were determined by HF/6-311++G(d,p) Time-Dependent method. The thermodynamic functions of the title molecule were also performed using the RHF and DFT methods. The restricted Hartree-Fock and density functional theory-based nuclear magnetic resonance (NMR) calculation procedure was also performed, and it was used for assigning the (13)C and (1)H NMR chemical shifts of Carbamazepine.

Transcriptional elongation of c-myb is regulated by NF-kappaB (p50/RelB).

High levels of c-myb expression are necessary for the proliferation of hematopoietic precursor cells whereas down-regulation of c-myb is required for terminal differentiation; this down-regulation occurs through a conditional block to transcriptional elongation in intron I. We previously observed that cAMP analogs prevented the late down-regulation of c-myb during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells and blocked differentiation; this correlated with the induction of NF-kappaB (p50/RelB) complexes which were shown to bind to NF-kappaB recognition sites flanking the transcriptional pause site of c-myb. We now selected stably-transfected MEL cells which overexpressed p50, RelB or both at levels similar to those induced by cAMP to determine whether these NF-kappaB proteins regulate c-myb expression in intact cells. We demonstrate that transcriptionally active NF-kappaB (p50/RelB) complexes, but not p50 or RelB alone, prevented the early and late down-regulation of c-myb mRNA and increased c-myb transcriptional elongation in HMBA-induced MEL cells. The increase in c-myb expression was sufficient to block erythroid differentiation and allow continuous proliferation of cells in the presence of HMBA. Steady-state c-myb mRNA levels in untreated cells were not affected by overexpression of NF-kappaB, suggesting that p50/RelB specifically modulated the efficiency of transcriptional attenuation during MEL cell differentiation.

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.

Regulation of the erythroid transcription factor NF-E2 by cyclic adenosine monophosphate-dependent protein kinase.

Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase-deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser(169); Ala substitution for Ser(169) resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore beta-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser (169)--> Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.

cAMP-induced NF-kappaB (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation.

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kappaB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers.

Nitric oxide-releasing agents and cGMP analogues inhibit murine erythroleukemia cell differentiation and suppress erythroid-specific gene expression: correlation with decreased DNA binding of NF-E2 and altered c-myb mRNA expression.

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.

Molecular cloning and characterization of a TAR-binding nuclear factor from T cells.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.

cAMP-dependent protein kinase is necessary for increased NF-E2.DNA complex formation during erythroleukemia cell differentiation.

When murine erythroleukemia (MEL) cells are induced to differentiate by hexamethylene bisacetamide (HMBA), erythroid-specific genes are transcriptionally activated; however, transcriptional activation of these genes is severely impaired in cAMP-dependent protein kinase (protein kinase A)-deficient MEL cells. The transcription factor NF-E2, composed of a 45-kDa (p45) and an 18-kDa (p18) subunit, is essential for enhancer activity of the globin locus control regions (LCRs). DNA binding of NF-E2 and alpha-globin LCR enhancer activity was significantly less in HMBA-treated protein kinase A-deficient cells compared to cells containing normal protein kinase A activity; DNA binding of several other transcription factors was the same in both cell types. In parental cells, HMBA treatment and/or prolonged activation of protein kinase A increased the amount of NF-E2.DNA complexes without change in DNA binding affinity; the expression of p45 and p18 was the same under all conditions. p45 and p18 were phosphorylated by protein kinase A in vitro, but the phosphorylation did not affect NF-E2.DNA complexes, suggesting that protein kinase A regulates NF-E2.DNA complex formation indirectly, e.g. by altering expression of a regulatory factor(s). Thus, protein kinase A appears to be necessary for increased NF-E2.DNA complex formation during differentiation of MEL cells and may influence erythroid-specific gene expression through this mechanism.

Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells.

Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.

Isolation and characterization of HL-60 cells resistant to nitroprusside-induced differentiation.

Sodium nitroprusside and sodium nitrite, which generate nitric oxide and increase the intracellular cGMP concentration, and 8-bromo-cGMP, a membrane-permeable cGMP analog, induce myelomonocytic differentiation of HL-60 cells (Boss, G. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7174-7178). We have selected HL-60 cells resistant to nitroprusside-induced differentiation as assessed by acquisition of the OKM-1 antigen, reduction of nitro blue tetrazolium, and morphologic maturation. The variant cells were also resistant to differentiation induced by sodium nitrite and two cGMP analogs but still differentiated in response to other inducing agents such as dimethyl sulfoxide and cAMP analogs and showed the same changes in c-myc and c-fos expression in response to the latter drugs as occurred in parental cells. We studied the early steps of the NO/cGMP signal transduction pathway in the variant cells and found that basal and nitroprusside-stimulated guanylate cyclase activity was similar in parental and variant cell extracts and that nitroprusside increased the intracellular cGMP concentration to the same extent in parental and variant cells. As part of these studies we found that HL-60 cells expressed only alpha 2 and beta 2 guanylate cyclase mRNA; the abundance of these two mRNA species was similar in parental and variant cells. Neither nitroprusside nor 8-bromo-cGMP changed the intracellular calcium concentration in parental or variant cells. The data suggest that the defect in the variant cells is after guanylate cyclase activation in the NO/cGMP transduction pathway and that the cGMP and cAMP transduction pathways operate independently in inducing differentiation of HL-60 cells.

Impairment of Photorespiratory Carbon Flow into Rubber by the Inhibition of the Glycolate Pathway in Guayule (Parthenium argentatum Gray).

Cut shoots of guayule (Parthenium argentatum Gray) were treated with four inhibitors of the glycolate pathway (alpha-hydroxypyridinemethanesulfonic acid; isonicotinic acid hydrazide, glycine hydroxamate, and amino-oxyacetate, AOA) in order to evaluate the role of photorespiratory intermediates in providing precursors for the biosynthesis of rubber. Photorespiratory CO(2) evolution in guayule leaves was severely inhibited by AOA. Application of each of the four inhibitors has resulted in a significantly decreased incorporation of (14)C into rubber fractions suggesting that the glycolate pathway is involved in the biosynthesis of rubber in guayule. However, the application of each of the glycolate pathway inhibitors showed no significant effect on photosynthetic CO(2) fixation in the leaves. The inhibitors individually also reduced the incorporation of labeled glycolate, glyoxylate, and glycine into rubber, while the incorporation of serine and pyruvate was not affected. The effective inhibition of incorporation of glycolate pathway intermediates in the presence of AOA was due to an inhibition of glycine decarboxylase and serine hydroxymethyltransferase. It is concluded that serine is a putative photorespiratory intermediate in the biosynthesis of rubber via pyruvate and acetyl coenzyme A.