The MC160 protein of the molluscum contagiosum virus dampens cGAS/STING-induced interferon-ß activation.

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign, persistent skin lesions. MCV encodes a variety of immune evasion molecules to dampen host immune responses. Two of these proteins are the MC159 and MC160 proteins. Both MC159 and MC160 contain two tandem death effector domains and share homology to the cellular FLIPs, FADD, and procaspase-8. MC159 and MC160 dampen several innate immune responses such as NF-κB activation and mitochondrial antiviral signaling (MAVS)-mediated induction of type 1 interferon (IFN). The type 1 IFN response is also activated by the cytosolic DNA sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Both cGAS and STING play a vital role in sensing a poxvirus infection. In this study, we demonstrate that there are nuanced differences between both MC160 and MC159 in terms of how the viral proteins modulate the cGAS/STING and MAVS pathways. Specifically, MC160 expression, but not MC159 expression, dampens cGAS/STING-mediated induction of IFN in HEK 293 T cells. Further, MC160 expression prevented the K63-ubiquitination of both STING and TBK1, a kinase downstream of cGAS/STING. Ectopic expression of the MC160 protein, but not the MC159 protein, resulted in a measurable decrease in the TBK1 protein levels as detected via immunoblotting. Finally, using a panel of MC160 truncation mutants, we report that the MC160 protein requires both DEDs to inhibit cGAS/STING-induced activation of IFN-ß. Our model indicates MC160 likely alters the TBK1 signaling complex to decrease IFN-ß activation at the molecular intersection of the cGAS/STING and MAVS signaling pathways.

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION: HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions.

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNß activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1 = R67A D69A; DED2 = R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

In vitro efficacy of 2,N-bisarylated 2-ethoxyacetamides against Plasmodium falciparum.

Investigation of a series of 2,N-bisarylated 2-ethoxyacetamides resulted in the identification of four inhibitors 5, 20, 24, 29 with single-digit micromolar in vitro efficacy against two drug-resistant Plasmodium falciparum strains. These compounds are analogs of structurally-related 1,3-bisaryl-2-propen-1-ones (chalcones), the latter showing efficacy in vitro but not in a malaria-infected mouse. The 2,N-bisarylated 2-ethoxyacetamides (e.g., 2, 5, 20) were shown to possess significantly greater stability in the presence of metabolizing enzymes than the corresponding 1,3-bisaryl-2-propen-1-ones (e.g., 1, 3, 18).

A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses.

To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR(-/-)) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. Importance: Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.

In vitro biotransformation, in vivo efficacy and pharmacokinetics of antimalarial chalcones.

4'-n-Butoxy-2,4-dimethoxy-chalcone (MBC) has been described as protecting mice from an otherwise lethal infection with Plasmodium yoelii when dosed orally at 50 mg/kg/dose, daily for 5 days. In contrast, we found that oral dosing of MBC at 640 mg/kg/dose, daily for 5 days, failed to extend the survivability of P. berghei-infected mice. The timing of compound administration and metabolic activation likely contribute to the outcome of efficacy testing in vivo. Microsomal digest of MBC yielded 4'-n-butoxy-4-hydroxy-2-methoxy-chalcone and 4'-(1-hydroxy-n-butoxy)-2,4-dimethoxy-chalcone. We propose that the latter will hydrolyze in vivo to 4'-hydroxy-2,4-dimethoxy-chalcone, which has greater efficacy than MBC in our P. berghei-infected mouse model and was detected in plasma following oral dosing of mice with MBC. Pharmacokinetic parameters suggest that poor absorption, distribution, metabolism and excretion properties contribute to the limited in vivo efficacy observed for MBC and its analogs.

Poxvirus MC160 protein utilizes multiple mechanisms to inhibit NF-kappaB activation mediated via components of the tumor necrosis factor receptor 1 signal transduction pathway.

Poxviruses express proteins that limit host immune responses to infection. For example, the molluscum contagiosum virus MC160 protein inhibits tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. This event correlates with MC160-induced IKK1 protein degradation, suggesting a mechanism for the above-mentioned phenotype. IKK1 is stabilized when it associates with the cellular heat shock protein 90 (Hsp90). Here, Hsp90 overexpression restored IKK1 levels in MC160-expressing cells, suggesting that MC160 competitively interacted with Hsp90. In support of this, further investigation showed that a mutant MC160 protein comprising only the C-terminal region (C protein) immunoprecipitated with Hsp90. In contrast, Hsp90 IP with a mutant MC160 protein consisting of only the N-terminal tandem death effector domains (DEDs) (N protein) was dramatically decreased. Since cells expressing either the N or C mutant MC160 protein remained similarly resistant to TNF-alpha-induced NF-kappaB activation, the N mutant protein probably utilized a different mechanism for inhibiting NF-kappaB. One likely mechanism for the N protein lies in its association with the DED-containing procaspase-8 protein, a cellular apoptosis precursor protein that regulates NF-kappaB activation. Here, IPs revealed that this association relied on the presence of the DED-containing N terminus of the MC160 protein but not the C-terminal portion. These interactions appear to have relevance with NF-kappaB activation, since the expression of the viral DEDs strongly inhibited procaspase-8-mediated NF-kappaB activation, an event not substantially altered by the C protein. Thus, the MC160 protein utilizes at least two distinct mechanisms for impeding NF-kappaB activation, association with Hsp90 to result in IKK1 protein degradation or interaction with procaspase-8.

Delayed Development of Spinal Subdural Hematoma Following Cranial Trauma: A Case Report and Review of the Literature.

BACKGROUND: Spinal subdural hematomas (SDHs) have been reported secondary to direct trauma or iatrogenic causes associated with coagulopathies. Spinal SDHs found after the development of acute intracranial SDHs, without any evidence of trauma to the spine, are extremely rare. In addition to this rare presentation, there is a lack of consensus regarding whether surgical decompression is the ideal treatment strategy. Depending on the extent of SDH within the spinal canal, surgical decompression may be difficult where diffuse hematoma within the intradural space requires multilevel decompression for treatment. CASE DESCRIPTION: A 46-year-old man initially presented with an acute cranial SDH following isolated head trauma. After a period of full recovery, he developed delayed lower extremity paraparesis secondary to the formation of a thoracolumbar SDH. This hematoma coincided with resolution of the cranial SDH and likely was due to redistribution of blood from the cranial subdural space into the spinal canal. Given the diffuse multilevel nature of the spread of hematoma and lack of a focal area of compression, he was managed conservatively. He demonstrated small signs of neurologic improvement over several days and regained considerable strength over the following several weeks. CONCLUSIONS: This report demonstrates a very rare occurrence of a traumatic intracranial SDH migrating into the thoracic and lumbar spine. This case also highlights that despite acute neurologic deficits, conservative management may be a feasible strategy that can result in recovery of neurologic function.

Selective inhibition of Pfmrk, a Plasmodium falciparum CDK, by antimalarial 1,3-diaryl-2-propenones.

The cyclin dependent protein kinases, Pfmrk and PfPK5, most likely play an essential role in cell cycle control and differentiation in Plasmodium falciparum and are thus an attractive target for antimalarial drug development. Various 1,3-diaryl-2-propenones (chalcone derivatives) which selectivity inhibit Pfmrk in the low micromolar range (over PfPK5) are identified. Molecular modeling shows a pair of amino acid residues within the Pfmrk active site which appear to confer this selectivity. Predicted interactions between the chalcones and Pfmrk correlate well with observed potency. Pfmrk inhibition and activity against the parasite in vitro correlate weakly. Several mechanisms of action have been suggested for chalcone derivatives and our study suggests that kinase inhibition may be an additional mechanism of antimalarial activity for this class of compounds.

Vegan Diet and Food Costs Among Adults With Overweight: A Secondary Analysis of a Randomized Clinical Trial.

This secondary analysis of a randomized clinical trial investigates the effects of a vegan diet on total food costs per day.

Poxviruses Utilize Multiple Strategies to Inhibit Apoptosis.

Cells have multiple means to induce apoptosis in response to viral infection. Poxviruses must prevent activation of cellular apoptosis to ensure successful replication. These viruses devote a substantial portion of their genome to immune evasion. Many of these immune evasion products expressed during infection antagonize cellular apoptotic pathways. Poxvirus products target multiple points in both the extrinsic and intrinsic apoptotic pathways, thereby mitigating apoptosis during infection. Interestingly, recent evidence indicates that poxviruses also hijack cellular means of eliminating apoptotic bodies as a means to spread cell to cell through a process called apoptotic mimicry. Poxviruses are the causative agent of many human and veterinary diseases. Further, there is substantial interest in developing these viruses as vectors for a variety of uses including vaccine delivery and as oncolytic viruses to treat certain human cancers. Therefore, an understanding of the molecular mechanisms through which poxviruses regulate the cellular apoptotic pathways remains a top research priority. In this review, we consider anti-apoptotic strategies of poxviruses focusing on three relevant poxvirus genera: Orthopoxvirus, Molluscipoxvirus, and Leporipoxvirus. All three genera express multiple products to inhibit both extrinsic and intrinsic apoptotic pathways with many of these products required for virulence.

The Molluscum Contagiosum Virus protein MC163 localizes to the mitochondria and dampens mitochondrial mediated apoptotic responses.

Apoptosis is a powerful host cell defense to prevent viruses from completing replication. Poxviruses have evolved complex means to dampen cellular apoptotic responses. The poxvirus, Molluscum Contagiosum Virus (MCV), encodes numerous host interacting molecules predicted to antagonize immune responses. However, the function of the majority of these MCV products has not been characterized. Here, we show that the MCV MC163 protein localized to the mitochondria via an N-terminal mitochondrial localization sequence and transmembrane domain. Transient expression of the MC163 protein prevented mitochondrial membrane permeabilization (MMP), an event central to cellular apoptotic responses, induced by either Tumor Necrosis Factor alpha (TNF-α) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). MC163 expression prevented the release of a mitochondrial intermembrane space reporter protein when cells were challenged with TNF-α. Inhibition of MMP was also observed in cell lines stably expressing MC163. MC163 expression may contribute to the persistence of MCV lesions by dampening cellular apoptotic responses.

5-Carba-pterocarpens: A new scaffold with anti-HCV activity.

The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5 µM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5 µM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.

The medicinal chemistry of botulinum, ricin and anthrax toxins.

The potential use of weapons of mass destruction (nuclear, biological or chemical) by terrorist organizations represents a major threat to world peace and safety. Only a limited number of vaccines are available to protect the general population from the medical consequences of these weapons. In addition there are major health concerns associated with a pre-exposure mass vaccination of the general population. To reduce or eliminate the impact of these terrible threats, new drugs must be developed to safely treat individuals exposed to these agents. A review of all therapeutic agents under development for the treatment of the illnesses and injuries that result from exposure to nuclear, biological or chemical warfare agents is beyond the scope of any single article. The intent here is to provide a focused review for medicinal and organic chemists of three widely discussed and easily deployed biological warfare agents, botulinum neurotoxin and ricin toxins and the bacteria Bacillus anthracis. Anthrax will be addressed because of its similarity in both structure and mechanism of catalytic activity with botulinum toxin. The common feature of these three agents is that they exhibit their biological activity via toxin enzymatic hydrolysis of a specific bond in their respective substrate molecules. A brief introduction to the history of each of the biological warfare agents is presented followed by a discussion on the mechanisms of action of each at the molecular level, and a review of current potential inhibitors under investigation.

A three-dimensional in silico pharmacophore model for inhibition of Plasmodium falciparum cyclin-dependent kinases and discovery of different classes of novel Pfmrk specific inhibitors.

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.

Oxindole-based compounds are selective inhibitors of Plasmodium falciparum cyclin dependent protein kinases.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Structure-activity relationship study of antimalarial indolo [2,1-b]quinazoline-6,12-diones (tryptanthrins). Three dimensional pharmacophore modeling and identification of new antimalarial candidates.

A widely applicable three-dimensional QSAR pharmacophore model for antimalarial activity was developed from a set of 17 substituted antimalarial indolo[2,1-b]quinazoline-6,12-diones (tryptanthrins) that exhibited remarkable in vitro activity (below 100 ng/mL) against sensitive and multidrug-resistant Plasmodium falciparum malaria. The pharmacophore, which contains two hydrogen bond acceptors (lipid) and two hydrophobic (aromatic) features, was found to map well onto many well-known antimalarial drug classes including quinolines, chalcones, rhodamine dyes, Pfmrk cyclin dependent kinase inhibitors, malarial FabH inhibitors, and plasmepsin inhibitors. The phamacophore allowed searches for new antimalarial candidates from multiconformer 3D databases and enabled custom designed synthesis of new potent analogues.

Methamphetamine and amphetamine isomer concentrations in human urine following controlled Vicks VapoInhaler administration.

Legitimate use of legal intranasal decongestants containing l-methamphetamine may complicate interpretation of urine drug tests positive for amphetamines. Our study hypotheses were that commonly used immunoassays would produce no false-positive results and a recently developed enantiomer-specific gas chromatography-mass spectrometry (GC-MS) procedure would find no d-amphetamine or d-methamphetamine in urine following controlled Vicks VapoInhaler administration at manufacturer's recommended doses. To evaluate these hypotheses, 22 healthy adults were each administered one dose (two inhalations in each nostril) of a Vicks VapoInhaler every 2 h for 10 h on Day 1 (six doses), followed by a single dose on Day 2. Every urine specimen was collected as an individual void for 32 h after the first dose and assayed for d- and l-amphetamines specific isomers with a GC-MS method with >99% purity of R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl derivatives and 10 µg/L lower limits of quantification. No d-methamphetamine or d-amphetamine was detected in any urine specimen by GC-MS. The median l-methamphetamine maximum concentration was 62.8 µg/L (range: 11.0-1,440). Only two subjects had detectable l-amphetamine, with maximum concentrations coinciding with l-methamphetamine peak levels, and always ≤ 4% of the parent's maximum. Three commercial immunoassays for amphetamines EMIT(®) II Plus, KIMS(®) II and DRI(®) had sensitivities, specificities and efficiencies of 100, 97.8, 97.8; 100, 99.6, 99.6 and 100, 100, 100%, respectively. The immunoassays had high efficiencies, but our first hypothesis was not affirmed. The EMIT(®) II Plus assay produced 2.2% false-positive results, requiring an enantiomer-specific confirmation.

Morphine and codeine concentrations in human urine following controlled poppy seeds administration of known opiate content.

Opiates are an important component for drug testing due to their high abuse potential. Proper urine opiate interpretation includes ruling out poppy seed ingestion; however, detailed elimination studies after controlled poppy seed administration with known morphine and codeine doses are not available. Therefore, we investigated urine opiate pharmacokinetics after controlled oral administration of uncooked poppy seeds with known morphine and codeine content. Participants were administered two 45 g oral poppy seed doses 8 h apart, each containing 15.7 mg morphine and 3mg codeine. Urine was collected ad libitum up to 32 h after the first dose. Specimens were analyzed with the Roche Opiates II immunoassay at 2000 and 300 µg/L cutoffs, and the ThermoFisher CEDIA(®) heroin metabolite (6-acetylmorphine, 6-AM) and Lin-Zhi 6-AM immunoassays with 10 µg/L cutoffs to determine if poppy seed ingestion could produce positive results in these heroin marker assays. In addition, all specimens were quantified for morphine and codeine by GC/MS. Participants (N=22) provided 391 urine specimens over 32 h following dosing; 26.6% and 83.4% were positive for morphine at 2000 and 300 µg/L GC/MS cutoffs, respectively. For the 19 subjects who completed the study, morphine concentrations ranged from <300 to 7522 µg/L with a median peak concentration of 5239 µg/L. The median first morphine-positive urine sample at 2000 µg/L cutoff concentration occurred at 6.6 h (1.2-12.1), with the last positive from 2.6 to 18 h after the second dose. No specimens were positive for codeine at a cutoff concentration of 2000 µg/L, but 20.2% exceeded 300 µg/L, with peak concentrations of 658 µg/L (284-1540). The Roche Opiates II immunoassay had efficiencies greater than 96% for the 2000 and 300 µg/L cutoffs. The CEDIA 6-AM immunoassay had a specificity of 91%, while the Lin-Zhi assay had no false positive results. These data provide valuable information for interpreting urine opiate results.

2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-nucleoside inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as inhibitors of HCV NS5B polymerase. Eleven compounds tested were found to inhibit HCV NS5B with IC50 values ranging between 19.8 and 64.9 µM. Compound 24 was the most active of this series with an IC50 of 5.6 µM. A number of these derivatives further exhibited strong inhibition against HCV 1b and 2a genotypes in cell based antiviral assays. Molecular docking analysis predicted that the thiazolidinone derivatives bind to the NS5B thumb pocket-II (TP-II). Our results suggest that further optimization of the thiazolidinone scaffold may be possible to yield new derivatives with improved enzyme- and cell-based activity.