Molecular mechanism of an antagonistic antibody against glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.

Anti-obesity effects of GIPR antagonists alone and in combination with GLP-1R agonists in preclinical models.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

Agonistic ß-Klotho antibody mimics fibroblast growth factor 21 (FGF21) functions.

Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. FGF21 functions through a receptor complex that consists of an FGF receptor (FGFR) and a co-receptor ß-Klotho. Here, we identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against ß-Klotho that mimics FGF21 function. The co-crystal structure of ß-Klotho KL1 domain in complex with 39F7 Fab revealed that the recognition of 39F7 is centered on Trp-295 of ß-Klotho in a FGF21 noncompetitive manner. KL1 adopts a (ß/α)8 TIM barrel fold which resembles that of ß-glycosylceramidase, but lacks molecular features for enzymatic activity, suggesting that KL1 functions as a scaffold protein instead. In vitro characterization demonstrated that, although 39F7 does not compete with FGF21, it is specific for ß-Klotho/FGFR1c activation. Furthermore, the agonistic activity of 39F7 required the full IgG molecule to be bivalent, suggesting that 39F7 functions by promoting receptor/co-receptor dimerization. Supported by negative stain EM analysis of full-length ß-Klotho, we propose a molecular model wherein the agonistic antibody 39F7 acts in a ß-Klotho- and FGFR1c-dependent manner, mimicking FGF21 activity. More importantly, 39F7 offers promising therapeutic potential in the axis of FGF21 signaling as an antibody therapy alternative to FGF21 analogs for treatment of metabolic diseases.

BacMam production of active recombinant lecithin-cholesterol acyltransferase: Expression, purification and characterization.

Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.

The high-resolution crystal structure of human LCAT.

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/ß hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.

Structure-assisted discovery of the first non-retinoid ligands for Retinol-Binding Protein 4.

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4-TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.

Molecular basis of sphingosine kinase 1 substrate recognition and catalysis.

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

Structure-guided design, synthesis, and evaluation of guanine-derived inhibitors of the eIF4E mRNA-cap interaction.

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

Genetic transformation of the codling moth, Cydia pomonella L., with piggyBac EGFP.

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.

Thermodynamic analysis of mRNA cap binding by the human initiation factor eIF4E via free energy perturbations.

Eukaryotic mRNAs are appended at the 5' end, with the 7-methylguanosine cap linked by a 5'-5'-triphosphate bridge to the first transcribed nucleoside (m7GpppX). Initiation of cap-dependent translation of mRNA requires direct interaction between the cap structure and the eukaryotic translation initiation factor eIF4E. Biophysical studies of the association between eIF4E and various cap analogs have demonstrated that m(7)GTP binds to the protein ca. -5.0 kcal/mol more favorably than unmethylated GTP. In this work, a thermodynamic analysis of the binding process between eIF4E and several cap analogs has been conducted using Monte Carlo (MC) simulations in conjunction with free energy perturbation (FEP) calculations. To address the role of the 7-methyl group in the eIF4E/m7GpppX cap interaction, binding free energies have been computed for m(7)GTP, GTP, protonated GTP at N(7), the 7-methyldeazaguanosine 5'-triphosphate (m(7)DTP), and 7-deazaguanosine 5'-triphosphate (DTP) cap analogs. The MC/FEP simulations for the GTP-->m(7)DTP transformation demonstrate that half of the binding free energy gain of m(7)GTP with respect to GTP can be attributed to favorable van der Waals interactions with Trp166 and reduced desolvation penalty due to the N(7) methyl group. The methyl group both eliminates the desolvation penalty of the N(7) atom upon binding and creates a larger cavity within the solvent that further facilitates the desolvation step. Analysis of the pair m(7)GTP-m(7)DTP suggests that the remaining gain in affinity is related to the positive charge created on the guanine moiety due to the N(7) methylation. The charge provides favorable cation-pi interactions with Trp56 and Trp102 and decreases the negative molecular charge, which helps the transfer from the solvent, a more polar environment, to the protein.

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol.

Proprotein convertase subtilisin kexin type 9 (PCSK9) has been shown to be involved in the regulation of extracellular levels of the low-density lipoprotien receptor (LDLR). Although PCSK9 is a subtilase, it has not been shown to degrade the LDLR, and its LDLR-lowering mechanism remains uncertain. Here we report the crystal structure of human PCSK9 at 2.3 A resolution. PCSK9 has subtilisin-like pro- and catalytic domains, and the stable interaction between these domains prevents access to PCSK9's catalytic site. The C-terminal domain of PCSK9 has a novel protein fold and may mediate protein-protein interactions. The structure of PCSK9 provides insight into its biochemical characteristics and biological function.

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the approximately 13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5' regulatory sequences.