Fibroblast growth factor 21 controls glycemia via regulation of hepatic glucose flux and insulin sensitivity.

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator shown to improve glycemic control. However, the molecular and functional mechanisms underlying FGF21-mediated improvements in glycemic control are not completely understood. We examined FGF21 effects on insulin sensitivity and glucose fluxes upon chronic (daily injection for 8 d) and acute (6 h infusion) administration in ob/+ and ob/ob mice. Results show that chronic FGF21 ameliorated fasting hyperglycemia in ob/ob mice via increased glucose disposal and improved hepatic insulin sensitivity. Acute FGF21 suppressed hepatic glucose production, increased liver glycogen, lowered glucagon, and improved glucose clearance in ob/+ mice. These effects were blunted in ob/ob mice. Neither chronic nor acute FGF21 altered skeletal muscle or adipose tissue glucose uptake in either genotype. In conclusion, FGF21 has potent glycemic effects caused by hepatic changes in glucose flux and improved insulin sensitivity. Thus, these studies define mechanisms underlying anti-hyperglycemic actions of FGF21 and support its therapeutic potential.

FGF21: a novel prospect for the treatment of metabolic diseases.

The increasing prevalence of metabolic diseases is alarming and highlights the need for more effective and safer therapies to treat these diseases. Recent evidence from several animal studies indicates that FGF21 induces numerous beneficial metabolic changes without apparent adverse effects. These results suggest that FGF21 could be a novel and attractive drug candidate for the treatment of cardiovascular disease, obesity and type 2 diabetes. The pharmacology of FGF21, molecular mechanisms contributing to the actions of this compound, and knowledge gaps to be addressed to allow further exploration of the therapeutic potential of this molecule are discussed in this review.

Fibroblast growth factor-21 as a therapeutic agent for metabolic diseases.

Fibroblast growth factor (FGF)-21 is a unique member of the FGF family, with several molecular characteristics that differ from classical FGFs and exhibiting a pharmacologic profile that includes a variety of metabolic responses in vitro and when tested in vivo in animal models. FGF21 represents a novel and attractive therapeutic agent for type 2 diabetes mellitus, because of its ability to modulate disease phenotype in preclinical settings without inducing any apparent adverse effects. Although FGF21 was discovered relatively recently, the understanding of its biology and therapeutic utility is rapidly evolving. A number of key metabolically linked molecules and pathways have been suggested to be involved in the mechanism of action of FGF21, depending on the specific target tissue/organ. Further research into these mechanisms should lead to important advances in the understanding of FGF21 biology and pave the way for novel therapeutic strategies. The specifics of FGF21 activities both in cell culture and in vivo, its potential as a target for diabetes, and insights into the molecular mechanisms of FGF21 metabolic actions will be discussed in this review.

FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.

Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.

The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21.

Fibroblast growth factor (FGF)-21 has been recently characterized as a potent metabolic regulator. Systemic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in genetically compromised diabetic rodents. Importantly, these effects were durable and did not come at the expense of weight gain, hypoglycemia, or mitogenicity. To explore the therapeutic properties of FGF-21 in a nongenetically modified primate species, and thus demonstrate the potential for efficacy in humans, we evaluated its bioactivity in diabetic nonhuman primates. When administered daily for 6 wk to diabetic rhesus monkeys, FGF-21 caused a dramatic decline in fasting plasma glucose, fructosamine, triglycerides, insulin, and glucagon. Of significant importance in regard to safety, hypoglycemia was not observed at any point during the study. FGF-21 administration also led to significant improvements in lipoprotein profiles, including lowering of low-density lipoprotein cholesterol and raising of high-density lipoprotein cholesterol, beneficial changes in the circulating levels of several cardiovascular risk markers/factors, and the induction of a small but significant weight loss. These data support the development of FGF-21 for the treatment of diabetes and other metabolic diseases.

Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression.

The detection of methamphetamine and other chemically related illicit drugs relies extensively on immunoassays. Here we report the cloning and affinity maturation of an anti-methamphetamine antibody which is being employed in the current commercial assays. An anti-methamphetamine scFv was cloned from hybridoma cells, expressed in bacteria and its affinity towards methamphetamine and N-ethylamphetamine (ethamphetamine) was determined by Surface Plasmon Resonance (SPR). The anti-methamphetamine scFv gene was subjected to random mutagenesis by error prone PCR and variants with improved affinity were isolated from the resulting library by a novel screening methodology termed Anchored Periplasmic Expression (APEx) [Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L., Rogers, G.K. (2004). Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.]. The isolated clones exhibited improved affinity to these illicit drugs, yet maintained low cross-reactivity to over-the-counter drugs. In addition, all clones displayed improved expression characteristics in Escherichia coli. The affinity improved scFv antibodies are thus likely to be useful in methamphetamine class immunodiagnostics.

Enhancing exposure of protein therapeutics.

Therapeutic proteins have made a major impact on medicine, with significant expansion in the past two decades. The medicinal attributes of these agents, particularly their efficacy and often their safety profile, make protein therapeutics attractive, despite the general necessity of invasive (parenteral) delivery. This perceived hurdle has been a primary component in limiting expansion of this class of drug therapies. Strategies that reduce the frequency of administration directly provide greater convenience to the patient, and potentially greater efficacy, that can yield a significant treatment advantage.:

FGF-21 as a novel metabolic regulator.

Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21-transgenic mice were viable and resistant to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.

Diversity in hapten recognition: structural study of an anti-cocaine antibody M82G2.

Antibodies against cocaine and other drugs of abuse are the basis for diagnostic tests for the presence of those drugs in human serum. The 1.7A resolution crystal structure of the anti-cocaine monoclonal antibody M82G2 in complex with cocaine is presented. This structure determination was undertaken to establish the stereochemical features in the antibody binding site that confer specificity for cocaine, and as part of an ongoing project to understand the rules that govern molecular recognition. The cocaine-binding site can be characterized topologically as a narrow groove on the protein surface. The antibody utilizes water-mediated hydrogen bonding, and cation-pi and stacking (pi-pi) interactions to provide specificity. Comparison with the previously published structure of the anti-cocaine antibody GNC92H2 shows that binding of a small ligand can be achieved in diverse ways, both in terms of a binding site structure/topology and protein-ligand interactions.

Medicinally useful proteins--enhancing the probability of technical success in the clinic.

Proteins that are normally present in organisms function to maintain the overall homeostatic balance of the living system. Although an understanding of the biology of these proteins is essential to determining their utility in the treatment of disease, the administration of wild-type forms of proteins in a therapeutic manner (often systemically and at concentrations much higher than those found in the healthy organism) can have the effect of inappropriate activation of cell types and compartments that can lead to an undesirable response. Modification of the sequence of the parent protein to yield selected biological properties can generate derivative versions (muteins) having an optimal therapeutic profile. In addition to consideration of the biological properties, protein therapeutics are typically not active when delivered orally. This can limit the frequency with which the therapeutic agent can be administered to the patient, exemplifying the need to understand the pharmacokinetic and pharmacodynamic relationships of the protein. The biophysical properties of the mutein (e.g., solubility and stability) must also be considered in this context. An additional consideration with therapeutic proteins, engineered or not, is the potential immune response resulting from sequence variation and/or exogenous presentation to the immune system. The composite application of these principles to therapeutic protein discovery and development drives the ultimate success of any given biopharmaceutical agent.

Anchoring a cationic ligand: the structure of the Fab fragment of the anti-morphine antibody 9B1 and its complex with morphine.

The crystal structures of an anti-morphine antibody 9B1 (to 1.6A resolution) and its complex with morphine (to 2.0 A resolution) are reported. The morphine-binding site is described as a shallow depression on the protein surface, an unusual topology for a high-affinity ( Ka approximately 10(9) M(-1)) antibody against a small antigen. The polar part of the ligand is exposed to solvent, and the cationic nitrogen atom of the morphine molecule is anchored at the bottom of the binding site by a salt-bridge to a glutamate side-chain. Additional affinity is provided by a double cation-pi interaction with two tryptophan residues. Comparison of the morphine complex with the structure of the free Fab shows that a domain closure occurs upon binding of the ligand.

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide.

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.

Therapeutic enhancement of IL-2 through molecular design.

A recombinant human IL-2 analog (rIL-2, Proleukin) is currently being evaluated for clinical benefit in HIV infected patients. It is approved for therapy of patients with metastatic melanoma and renal cell carcinoma. Treatment of cancer patients with rIL-2 results in durable responses but is associated with life-threatening toxicity, which limits its use to patients in relatively good health. Antitumor efficacy associated with rIL-2 therapy are hypothesized to be mediated by distinct types of cells that express structurally different forms of the IL-2 receptor. This hypothesis suggests that it might be possible to engineer an IL-2 variant addressing the risks associated with the therapeutic use of IL-2. In this article, we review the clinical experience with IL-2 and its analogs, the evidence that different IL-2 receptors may dissociate efficacy and toxicity, and describe the generation of a novel IL-2 variant with the potential for a superior therapeutic index.