Patti A. Longo

Glucose and Weight Control in Mice with a Designed Ghrelin O-Acyltransferase Inhibitor

Metabolism Without Modification Obesity-associated metabolic disease has rapidly become a public health priority in the developed world and is being addressed through prevention strategies aimed at lifestyle changes and through pharmacological approaches. Barnett et al. (p. 1689, published online 18 November) designed a drug that inhibits the action of ghrelin, a circulating peptide hormone that increases fat mass and food intake. The drug, a bisubstrate analog called GO-CoA-Tat, is a selective antagonist of ghrelin O-acyltransferase (GOAT), an enzyme that catalyzes a posttranslational modification that is essential for ghrelin activity. Injection of GO-CoA-Tat into wild-type mice on a high-fat diet improved glucose tolerance and reduced weight gain, probably through changes in metabolic activity. Because GO-CoA-Tat is a peptide-based drug that requires repeated injection, it is unsuitable for clinical use, but GOAT does represent a potentially valuable target for future drug development efforts in metabolic disease. A drug inhibiting the activation of ghrelin, a peptide that promotes weight gain, has beneficial metabolic effects in mice. Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser3, an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.

Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences.

A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (P < 0.000001), but instability tended to be more widespread in SRSCCa (P = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline hMSH2 mutation (P = 0.0007). The prevalence of K-ras proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline hMSH2 mutation had codon 13 mutation (P = 0.02), and two other HNPCCa had multiple K-ras mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI (P = 0.0004 and P = 0.0001, respectively). Frameshift mutation of the transforming growth factor beta type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the E2F-4 transcription factor gene was more common in HNPCCa of patients with germline hMSH2 mutation than in those with germline bMLH1 mutation (100% (8/8) versus 40% (2/5), P = 0.04), and mutation of the Bax proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) versus 13% (2/15), P = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, ras, E2F-4, and Bax genes), but not others (eg, transforming growth factor beta type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.

A single ligand is sufficient to activate EGFR dimers

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions.

Mechanistic insights into the activation of oncogenic forms of EGF receptor.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly activated by mutation in non–small cell lung cancer. The mechanism of this oncogenic activation is not completely understood, but in contrast to that of the wild-type EGFR, it is proposed to be independent of kinase domain dimerization. Mechanistic studies on EGFR have mainly relied on cell-based assays or isolated kinase domain measurements. Here we show, using purified, near full-length human EGFR proteins (tEGFRs), that two oncogenic mutants are fully active independently of EGF and highly resistant to the therapeutic and endogenous inhibitors cetuximab, lapatinib and MIG6. Based on the pattern of inhibition and the effects of additional asymmetric kinase dimer interface mutations, we propose that these oncogenic EGFR mutants drive and strongly depend on the formation of the asymmetric kinase dimer for activation, which has implications for drug design and cancer treatment strategies.

In vitro enzymatic characterization of near full length EGFR in activated and inhibited states.

The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is approximately 500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.