The Panitumumab EGFR Complex Reveals a Binding Mechanism That Overcomes Cetuximab Induced Resistance.

Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R) confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation.

Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity.

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Role of passive and adaptive immunity in influencing enterocyte-specific gene expression.

Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the influence that adaptive (AI) and passive (PI) immunity have in controlling their expression has not been evaluated. In this study, we tested the hypothesis that both PI and AI influenced enterocyte gene expression by developing a breeding scheme that used T and B cell-deficient recombination-activating gene (RAG) mice. RNA was isolated from the liver and proximal/distal small intestine at various ages, and the steady-state levels of six different transcripts were evaluated by RNase protection assay. In wild-type (WT) pups, all transcripts [Fc receptor of the neonate (FcRn), polymeric IgA receptor (pIgR), GLUT5, lactase-phlorizin hydrolase (lactase), apical sodium-dependent bile acid transporter (ASBT), and Na+/glucose cotransporter (SGLT1)] studied were developmentally regulated at the time of weaning, and all transcripts except ASBT had the highest levels of expression in the proximal small intestine. In WT suckling pups reared in the absence of PI, pIgR mRNA levels were increased 100% during the early phase of development. In mice lacking AI, the expression of pIgR and lactase were significantly attenuated, whereas FcRn and GLUT5 levels were higher compared with WT mice. Finally, in the absence of both passive and active immunity, expression levels of pIgR and lactase were significantly lower than similarly aged WT mice. In summary, we report that the adaptive and passive immune status of mice influences steady-state mRNA levels of several important, developmentally regulated enterocyte genes during the suckling and weaning periods of life.

Multiple transcription factors in 5'-flanking region of human polymeric Ig receptor control its basal expression.

The polymeric Ig receptor (pIgR) is a critical component of the mucosal immune system and is expressed in largest amounts in the small intestine. In this study, we describe the initial characterization of the core promoter region of this gene. Expression of chimeric promoter-reporter constructs was supported in Caco-2 and HT-29 cells, and DNase I footprint analysis revealed a large protein complex within the core promoter region. Site-directed mutagenesis experiments determined that elements within this region serve to either augment or repress basal activity of the human pIgR promoter. Band shift assays of overlapping oligonucleotides within the core promoter identified eight distinct complexes; the abundance of most complexes was enhanced in post-confluent cells. In summary, we report the characterization of the human pIgR promoter and the essential role that eight different nuclear complexes have in controlling basal expression of this gene in Caco-2 cells.