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Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

The combination of the β-lactam tebipenem and the β-lactamase inhibitor avibactam shows potent bactericidal activity against Mycobacterium abscessus in vitro . Here, we report that the combination of the respective oral prodrugs tebipenem-pivoxil and avibactam ARX-1796 showed efficacy in a mouse model of M. abscessus lung infection.

The sensitivity of Mycobacterium tuberculosis , the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis , which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

The sensitivity of

In the course of optimizing a novel indazole sulfonamide series that inhibits β-ketoacyl-ACP synthase (KasA) of Mycobacterium tuberculosis, a mutagenic aniline metabolite was identified. Further lead optimization efforts were therefore dedicated to eliminating this critical liability by removing the embedded aniline moiety or modifying its steric or electronic environment. While the narrow SAR space against the target ultimately rendered this goal unsuccessful, key structural knowledge around the binding site of this underexplored target for TB was generated to inform future discovery efforts.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill

[Image: see text] In this study, a series of 49 five-membered heterocyclic compounds containing either a pyridine- or a pyrrole-type nitrogen were synthesized and tested against Mycobacterium tuberculosis. Among them, only the 1,3,5-trisubstituted pyrazoles 5–49 exhibited minimum inhibitory concentration values in the low micromolar range, and some also exhibited an improved physicochemical profile without cytotoxic effects. Three pyrazoles were subjected to an animal tuberculosis efficacy model, and compound 6 induced a statistically significant difference in lung bacterial counts compared with untreated mice. Moreover, to determine the target of this series, resistors were generated, and whole genome sequencing revealed mutations in the mmpL3 gene.

Isoniazid (INH) remains one of the cornerstones of antitubercular chemotherapy for drug-sensitive strains of M. tuberculosis bacteria. However, the increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains containing mutations in the KatG enzyme, which is responsible for the activation of INH into its antitubercular form, have rendered this drug of little or no use in many cases of drug-resistant tuberculosis. Presented herein is a novel family of antitubercular direct NADH-dependent 2-trans enoyl-acyl carrier protein reductase (InhA) inhibitors based on an N-benzyl-4-((heteroaryl)methyl)benzamide template; unlike INH, these do not require prior activation by KatG. Given their direct InhA target engagement, these compounds should be able to circumvent KatG-related resistance in the clinic. The lead molecules were shown to be potent inhibitors of InhA and showed activity against M. tuberculosis bacteria. This new family of inhibitors was found to be chemically tractable, as exemplified by the facile synthesis of analogues and the establishment of structure-activity relationships. Furthermore, a co-crystal structure of the initial hit with the enzyme is disclosed, providing valuable information toward the design of new InhA inhibitors for the treatment of MDR/XDR tuberculosis.

Society urgently needs new, effective medicines for the treatment of tuberculosis. To kick-start the required hit-to-lead campaigns, the libraries of pharmaceutical companies have recently been evaluated for starting points. The GlaxoSmithKline (GSK) library yielded many high-quality hits, and the associated data were placed in the public domain to stimulate engagement by the wider community. One such series, the spiro compounds, are described here. The compounds were explored by a combination of traditional in-house research and open source methods. The series benefits from a particularly simple structure and a short associated synthetic chemistry route. Many members of the series displayed striking potency and low toxicity, and highly promising in vivo activity in a mouse model was confirmed with one of the analogues. Ultimately the series was discontinued due to concerns over safety, but the associated data remain public domain, empowering others to resume the series if the perceived deficiencies can be overcome.

Antifolates are widely used to treat several diseases but are not currently used in the first-line treatment of tuberculosis, despite evidence that some of these molecules can target Mycobacterium tuberculosis (Mtb) bacilli in vitro. To identify new antifolate candidates for animal-model efficacy studies of tuberculosis, we paired knowledge and tools developed in academia with the infrastructure and chemistry resources of a large pharmaceutical company. Together we curated a focused library of 2508 potential antifolates, which were then tested for activity against live Mtb. We identified 210 primary hits, confirmed the on-target activity of potent compounds, and now report the identification and characterization of 5 hit compounds, representative of 5 different chemical scaffolds. These antifolates have potent activity against Mtb and represent good starting points for improvement that could lead to in vivo efficacy studies.

One way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series of Mycobacterium tuberculosis gyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 μM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 μM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 μM and 132 μM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection ...

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC ( T >MIC ) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.

Objective Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc. Methods The expression of messenger RNA (mRNA) for different C-C and C-X-C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP-1) protein production was analyzed by enzyme-linked immunosorbent assay. The chemotactic effect of fibroblast-derived MCP-1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) activation in fibroblasts was studied by electromobility shift analysis. MCP-1 expression in SSc skin sections was studied by immunohistochemistry. Results Among all chemokine genes studied, only MCP-1 and interleukin-8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP-1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP-1 production was associated with higher chemotactic activity for monocytic cells. Increased NF-κB or AP-1 activation was not responsible for the constitutive overexpression of MCP-1 by SSc fibroblasts. In SSc skin sections, MCP-1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin. Conclusion SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP-1 in vitro. MCP-1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

In HIV-infected T lymphocytes, NF-kappaB/Rel transcription factors are major elements involved in the activation of LTR-dependent transcription from latency. Most NF-kappaB heterodimer p65/p50 is sequestered as an inactive form in the cytoplasm of resting T lymphocytes via its interaction with I kappaB inhibitors. In these cells, both absolute HIV latency and low level ongoing HIV replication have been described. These situations could be related to differences in the balance between NF-kappaB and I kappaB alpha ratio. Actually, control of I kappaB alpha by cellular factors such as Murr-1 plays a critical role in maintaining HIV latency in unstimulated T lymphocytes. Formerly, our group demonstrated the presence of nuclear I kappaB alpha in T cells after PMA activation. Now we attempt to determine the dynamics of NF-kappaB/I kappaB alpha nucleocytosolic transport in absence of activation as a mechanism to explain both the maintenance of latency and the existence of low level ongoing HIV replication in resting CD4+ T lymphocytes.We show that the inhibition of the nuclear export by leptomycin B in resting CD4+ T cells resulted in nuclear accumulation of both I kappaB alpha and p65/RelA, as well as formation of NF-kappaB/I kappaB alpha complexes. This proves the existence of a rapid shuttling of I kappaB alpha between nucleus and cytosol even in absence of cellular activation. The nuclear accumulation of I kappaB alpha in resting CD4+ T lymphocytes results in inhibition of HIV-LTR dependent transcription as well as restrains HIV replication in CD4+ T lymphocytes. On the other hand, basal NF-kappaB activity detected in resting CD4+ T lymphocytes was related to low level HIV replication in these cells.

Objective The expansion of an aggressive population of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) synovium occurs despite their expression of functional death receptors and exposure to death receptor ligands. FLS can survive Fas challenge because of the constitutive expression of FLIP apoptosis inhibitor. We investigated whether Fas signaling plays a pathogenetic role by activating a nonapoptotic proinflammatory program in RA FLS. Methods Cultured RA FLS were stimulated with an agonistic anti-Fas antibody in the presence or absence of the caspase inhibitor Z-VAD-FMK or after RNA interference with a short hairpin RNA expression plasmid directed against FLIP. NF-κB and activator protein 1 (AP-1) activation was studied by electrophoretic mobility shift assays and p65 immunofluorescence analysis, and expression of messenger RNA (mRNA) for monocyte chemoattractant protein 1, interleukin-8, IκBα, and matrix metalloproteinases (MMPs) 1, 9, and 13 was examined by reverse transcription–polymerase chain reaction. Chemotactic activity of Fas-activated FLS–conditioned media was studied in Transwell migration assays. Results Fas stimulation activated NF-κB and AP-1, and this response required caspase activity, since Z-VAD-FMK inhibitor precluded it. FLIP was processed to p43 protein after Fas stimulation in a caspase-dependent manner, and inhibition of FLIP expression resulted in reduced Fas-triggered NF-κB activation. Fas stimulation increased expression of mRNA for IκBα, MMPs, and chemokines, and Fas-activated RA FLS displayed increased chemotactic activity for monocytic cells. Conclusion Fas triggering may contribute to the proinflammatory features of RA FLS by activating NF-κB and AP-1 and by expression of relevant target genes, such as MMPs and chemokines. Fas proinflammatory signaling is dependent upon caspase activity and FLIP expression. These data implicate FLIP as a potentially important molecular switch that turns the Fas signaling away from apoptosis and toward induction of a proinflammatory phenotype in RA FLS.

Background Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. Methods Samples of blood from Plasmodium yoelii–infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. Results The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. Conclusions The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria. © 2005 Wiley-Liss, Inc.

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase., This investigation was supported by research grants BMC2002- 03246 and BIO2000-0340-P4 from Spain and QLK2-CT2002-00954 from the European Union to M.E., by grants SAF2000-0028 and FIPSE to J.A., and by the Fundación Ramón Areces (P.G.-P.). H.N. is supported by a grant from the Human Frontier Science Program. M.A.G. and J.G. were supported by fellowships from the Ministerio de Ciencia y Tecnología, Madrid, Spain.

Sialoadhesin (sheep erythrocyte receptor, SER) is a macrophage-restricted adhesion molecule that binds certain sialylated ligands. It is borne by bone marrow stromal macrophages, promoting the interaction with developing myeloid cells, and by a subset of tissue macrophages involved in antigen presentation and activation of tumor-reactive T cells. The expression of sialoadhesin on SER+ macrophages is not constitutive but requires the continuous supply of a sialoadhesin-inducing serum factor. Tumor growth is often associated with marked alterations of myelopoiesis and impairment of T cell activation; yet the expression of sialoadhesin in macrophages derived from tumor bearers has not been addressed. The aim of this study was to assess whether Ehrlich tumor (ET) – a murine mammary carcinoma – growth may modify the sialoadhesin expression by bone marrow macrophages and/or sialoadhesin-inducing activity in ET-bearing sera. Moreover, putative functional sialoadhesin inhibitors produced by ET cells were tested. The results indicate that bone marrow cells from ET bearers show a seven- to eight-fold decrease in SER+ cells as detected by flow cytometry. This is accompanied by an overall decrease in sheep erythrocyte binding to tumor-bearer-derived bone marrow cells, but also by lower numbers of plastic-adherent cells. Functional sialoadhesin expression is preserved at the single-cell level and no inhibitors are found in ET-bearing sera or ET cell culture supernatants. Tumor progression does not impair the sialoadhesin-inducing activity of ET-bearing sera, or the ability of SER− macrophages (e.g. peritoneal macrophages) to respond to such an induction. In conclusion, while SER+ macrophages are greatly decreased in bone marrow from ET bearers, this is not due to a down-regulation of sialoadhesin expression, nor to an impairment of sialoadhesin-inducing factor or to the presence of sialoadhesin-binding moieties of tumor origin, but, more likely, to a decrease of fully mature macrophages.

Induction of HIV expression through lymphocyte activation has been proposed as a strategy to purge latent reservoirs. Prostratin is a non-tumourogenic phorbol ester that delays HIV replication in vitro, but paradoxically activates HIV expression in latently infected cells. To get a better insight into the mechanisms of action of prostratin, we have analysed the effect of prostratin on HIV activation and HIV receptor and coreceptors’ surface expression in human lymphocytes. Peripheral blood mononuclear cells (PBMCs) were transfected with luciferase expression constructs under the control of wild type HIV-long terminal repeat (LTR) and consensus sequences for transcription factors involved in HIV-LTR transactivation (NF-κB, SP1, NFAT). Prostratin stimulates transactivation of LTR vectors, κB- and SP-1-driven luciferase constructs. In another set of experiments, PBMCs were transfected with a full-length infectious viral clone. Prostratin induced HIV transcription and viral expression as detected by luciferase activity in cellular extracts and p24 levels in culture supernatants, respectively. Expression of the HIV coreceptors CCR5 and CXCR4 was decreased by prostratin and, concomitantly, prostratin inhibited the infection of PBMCs with R5 and X4 strains. However, prostratin did not inhibit infection with a pseudotyped viral clone that enters into the cells independently of HIV receptors. These results help to explain the paradoxical effects of prostratin. On one hand, prostratin induces HIV activation in latently infected cells through the induction of NF-κB and Sp1. On the other hand, strong and persistent downregulation of HIV receptors decreases infection of new targets and delays HIV propagation. These data support the potential use of prostratin to activate HIV from latency and purge viral reservoirs.

Molluscum contagiosum virus (MCV) is a human poxvirus that causes abnormal proliferation of epithelial cells. MCV encodes specific molecules to control host defences, such as MC159L, which as previously shown prevents apoptosis induced by death receptors. However, unlike most poxviruses, MCV lacks a homologue to the E3L and K3L proteins of vaccinia virus, which are involved in the control of the key antiviral and pro-apoptotic dsRNA-dependent protein kinase, PKR. In this study, we analysed the relationship of MC159L to PKR. We found that MC159L is not a direct inhibitor of PKR since it does not associate with PKR and cannot block PKR-induced phosphorylation of eIF-2α. However, expression of MC159L inhibits apoptosis triggered by PKR through death receptor-mediated pathways. In addition, MC159L inhibits NF-κB activation induced in response to PKR. Expression of MC159L cannot counteract the PKR-mediated antiviral action in the context of a poxvirus infection, despite its ability to affect these signalling events. These findings show that MC159L is able to interfere with downstream events triggered by PKR in the absence of a direct physical interaction, and assign a role to MC159L in the control of some PKR-mediated biological effects.