Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.

Human glioblastoma cells secrete a peptide, termed glioblastoma‐derived T cell suppressor factor (G‐TsF), which has suppressive effects on interleukin‐2‐dependent T cell growth. As shown here, complementary DNA for G‐TsF reveals that G‐TsF shares 71% amino acid homology with transforming growth factor‐beta (TGF‐beta). In analogy to TGF‐beta it is apparently synthesized as the carboxy‐terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino‐acid‐long mature form of G‐TsF. Comparison of the amino‐terminal sequence of G‐TsF with that of porcine TGF‐beta 2 and bovine cartilage‐inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G‐TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharidestimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFI-KELRVIESGPHCANTEIIVKLSDGRELCLDP-KENWVQRVVEKFLKRAENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino-and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

We have developed standardized procedures and practices for infection of SCID-hu Thy/Liv mice with human immunodeficiency virus type 1 for the prophylactic administration of antiviral compounds and for evaluation of the antiviral effect in vivo. Endpoint analyses included quantitation of viral load by intracellular p24 enzyme-linked immunosorbent assay, DNA PCR for the presence of proviral genomes, flow cytometry to measure the representation of CD4 ؉ and CD8 ؉ cells, and cocultivation for the isolation of virus. Efficacy tests in this model are demonstrated with the nucleoside analogs zidovudine and dideoxyinosine and with the nonnucleoside reverse transcriptase inhibitor nevirapine. This small-animal model should be particularly useful in the preclinical prioritization of lead compounds within a common chemical class, in the evaluation of alternative in vivo dosing regimens, and in the determination of appropriate combination therapy in vivo.