Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

Proper nucleosides availability is crucial for the proliferation of living entities (eukaryotic cells, parasites, bacteria, and virus). Accordingly, the uses of inhibitors of the de novo nucleosides biosynthetic pathways have been investigated in the past. In the following we have focused on dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine nucleosides biosynthetic pathway. We first described the different types of enzyme in terms of sequence, structure, and biochemistry, including the reported bioassays. In a second part, the series of inhibitors of this enzyme along with a description of their potential or actual uses were reviewed. These inhibitors are indeed used in medicine to treat autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (leflunomide and teriflunomide) and have been investigated in treatments of cancer, virus, and parasite infections (i.e., malaria) as well as in crop science.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in nucleotide biosynthesis studied as an important therapeutic target and its complex functioning in vivo is still puzzling and debated. Here, we highlight the structural basis for the regulation of IMPDHs by MgATP. Our results demonstrate the essential role of the CBS tandem, conserved among almost all IMPDHs. We found that Pseudomonas aeruginosa IMPDH is an octameric enzyme allosterically regulated by MgATP and showed that this octameric organization is widely conserved in the crystal structures of other IMPDHs. We also demonstrated that human IMPDH1 adopts two types of complementary octamers that can pile up into isolated fibers in the presence of MgATP. The aggregation of such fibers in the autosomal dominant mutant, D226N, could explain the onset of the retinopathy adRP10. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or macromolecular assembly.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6‐phosphate signal by two receptors: the cation‐independent mannose 6‐phosphate/insulin‐like growth factor II receptor (CI‐MPR) and the cation‐dependent mannose 6‐phosphate receptor (CD‐MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD‐MPR‐deficient mice with Thp mice that carry a CI‐MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI‐MPR, like those lacking the CD‐MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6‐phosphate binding proteins in mammalian cells.

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPRs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly. MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires two distinct determinants at the carboxyl terminus of the CD-MPR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.

No abstract

We present an improved version of the program LEA developed to design organic molecules. Rational drug design involves finding solutions to large combinatorial problems for which an exhaustive search is impractical. Genetic algorithms provide a tool for the investigation of such problems. New software, called LEA3D, is now able to conceive organic molecules by combining 3D fragments. Fragments were extracted from both biological compounds and known drugs. A fitness function guides the search process in optimizing the molecules toward an optimal value of the properties. The fitness function is build up by combining several independent property evaluations, including the score provided by the FlexX docking program. One application in de novo drug design is described. The example makes use of the structure of Mycobacterium tuberculosis thymidine monophosphate kinase to generate analogues of one of its natural substrates. Among 22 tested compounds, 17 show inhibitory activity in the micromolar range.

We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3Ј-azido-3Ј-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKmt. We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKmt. This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials.Keywords: Tuberculosis; circular dichroism; fluorescence spectroscopy; molecular modeling; structurefunction relationship Mycobacterium tuberculosis, the causative agent of tuberculosis, is the leading cause of death from infectious agents. Diverse factors, the most important being the acquired immunodeficiency syndrome (AIDS) epidemics, have provoked a resurgence of this disease in industrialized countries. In 1996, the World Health Organization (WHO) declared tuberculosis to be a global emergency, as multidrugresistant strains recently have emerged (for review, see Barry and Mdluli [1996] and Cole [1994]). Because the bacille Calmette-Guérin (BCG) vaccine efficacy is a subject of controversy, the search for new targets to obtain more effective drugs to control the spread of tuberculosis is a priority. In this respect, we have focused our attention on thymidylate kinase of M. tuberculosis (TMPKmt) for several reasons: (1) The enzyme, which phophorylates dTMP to dTDP, is essential for DNA synthesis in vivo as has been demonstrated with the cdc8 mutant in Saccharomyces cerevisiae. The CDC8 protein, the thymidylate kinase from yeast (TMPKy) (Jong et al. 1984;Sclafani and Fangman 1984), is a cell-cycle-regulated protein (White et al. 1987