SignificanceMutants of RAS are major oncogenes and occur in many human cancers, but efforts to develop drugs that directly inhibit the corresponding constitutively active RAS proteins have failed so far. We therefore focused on SOS1, the guanine nucleotide exchange factor (GEF) and activator of RAS. A combination of high-throughput and fragment screening resulted in the identification of nanomolar SOS1 inhibitors, which effectively down-regulate active RAS in tumor cells. In cells with wild-type KRAS, we observed complete inhibition of the RAS-RAF-MEK-ERK pathway. In a mutant KRAS cell line, SOS1 inhibition resulted in a reduction of phospho-ERK activity by 50%. Together, the data indicate that inhibition of GEFs may represent a viable approach for targeting RAS-driven tumors.

Purpose: The effects of pan^histone deacetylase (HDAC) inhibitors on cancer cells have shown that HDACs are involved in fundamental tumor biological processes such as cell cycle control, differentiation, and apoptosis. However, because of the unselective nature of these compounds, little is known about the contribution of individual HDAC family members to tumorigenesis and progression. The purpose of this study was to evaluate the role of individual HDACs in neuroblastoma tumorigenesis. Experimental Design: We have investigated the mRNA expression of all HDAC1-11 family members in a large cohort of primary neuroblastoma samples covering the full spectrum of the disease. HDACs associated with disease stage and survival were subsequently functionally evaluated in cell culture models. Results: Only HDAC8 expression was significantly correlated with advanced disease and metastasis and down-regulated in stage 4S neuroblastoma associated with spontaneous regression. High HDAC8 expression was associated with poor prognostic markers and poor overall and event-free survival. The knockdown of HDAC8 resulted in the inhibition of proliferation, reduced clonogenic growth, cell cycle arrest, and differentiation in cultured neuroblastoma cells. The treatment of neuroblastoma cell lines as well as short-term-culture neuroblastoma cells with an HDAC8-selective small-molecule inhibitor inhibited cell proliferation and clone formation, induced differentiation, and thus reproduced the HDAC8 knockdown phenotype. Global histone 4 acetylation was not affected by HDAC8 knockdown or by selective inhibitor treatment. Conclusions: Our data point toward an important role of HDAC8 in neuroblastoma pathogenesis and identify this HDAC family member as a specific drug target for the differentiation therapy of neuroblastoma.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N -[(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (␤␣) 8-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (␤␣) 8-barrel enzymes are very suitable for the design of new catalytic activities.E nzymes of contemporary metabolic pathways are generally specific and efficient biocatalysts. They can be categorized into a limited number of families, the members of which share similar reaction mechanisms, folds, or both (1). This leads to the idea that the members of a given enzyme family are evolutionarily related. In principle, two different evolutionary scenarios can be envisioned. New catalytic functions of enzymes could have evolved by changing the chemistry of catalysis, while retaining the binding capacity for a common ligand. This idea of retrograde evolution (2) was recently supported by the successful interconversion of the catalytic activity of two enzymes from tryptophan biosynthesis, which catalyze successive reactions in the pathway and therefore bind the same ligand (3). Alternatively, new catalytic functions could have evolved by retaining the chemistry of catalysis, while changing the substrate specificity (1). Along these lines, the patchwork model of enzyme evolution (4) postulates that ancestral enzymes were relatively unspecific and therefore were capable of catalyzing chemically similar reactions in different metabolic pathways. Genes encoding these enzymes would have duplicated in the course of evolution and would have subsequently specialized by diversification. NЈ-[(5Ј-Phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24) constitute a pair of similar enzymes, which are involved in the biosynthesis of the amino acids histidine and tryptophan, respectively (5, 6). Both HisA and TrpF catalyze an Amadori rearrangement, which is the irreversible isomerization of an aminoaldose to an aminoketose (Fig. 1). Also, despite the lack of detectable amino acid sequence similarity, HisA and TrpF belong to the same structural family of (␤␣) 8 -barrels (7, 8), which is the most frequent fold among single-dom...