Discovery of Truncated Cyclic Peptides Targeting an Induced-Fit Pocket on PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDL-R) degradation. We previously identified and optimized 13-mer cyclic peptides that bind to a novel, induced-fit pocket adjacent to the binding interface of PCSK9 and LDL-R and effectively disrupted the PCSK9/LDL-R protein-protein interaction (PPI) both in vitro and in vivo. However this series of large cyclic peptides required charged groups for function and lacked oral bioavailability in rodents. We describe herein multiple structure-based modifications to these original peptides to yield truncated, neutral molecules with full PPI function in both biochemical and cellular assays. In parallel, new mRNA-peptide display screens identified non-functional 8- and 9-mer compounds which ligand the induced-fit pocket in a distinct manner. Taken together, these studies indicate multiple directions to reduce the size and complexity of this peptide class toward a true small molecule oral agent.

Clearance of plasma PCSK9 via the asialoglycoprotein receptor mediated by heterobifunctional ligands.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting the degradation of hepatic LDL receptors (LDLRs). Current therapeutic approaches use antibodies that disrupt PCSK9 binding to LDLR to reduce circulating LDL-C concentrations or siRNA that reduces PCSK9 synthesis and thereby levels in circulation. Recent reports describe small molecules that, like therapeutic antibodies, interfere with PCSK9 binding to LDLR. We report an alternative approach to decrease circulating PCSK9 levels by accelerating PCSK9 clearance and degradation using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, demonstrate binding in vitro and accelerated PCSK9 clearance in vivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK.

Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor α (TNFα) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.

Dual-acting peptide with prolonged glucagon-like peptide-1 receptor agonist and glucagon receptor antagonist activity for the treatment of type 2 diabetes.

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9-39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

Discovery of an Orally Bioavailable Benzimidazole Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor That Suppresses Body Weight Gain in Diet-Induced Obese Dogs and Postprandial Triglycerides in Humans.

Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9 was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. 9 has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans.

Engineering of a VPAC2 receptor peptide agonist to impart dipeptidyl peptidase IV stability and enhance in vivo glucose disposal.

VPAC2P-PEG is a VPAC2 receptor agonist peptide that acts as a glucose-dependent insulin secretagogue. Proteolysis by DPPIV may contribute to the in vivo clearance of VPAC2P-PEG. Here, the N-terminus of VPAC2P-PEG is modified by N-terminal acetylation to impart DPPIV resistance. The acetylated peptide, Ac-VPAC2P-PEG, is a selective and potent VPAC2 agonist, resistant to DPPIV proteolysis, and exhibits substantially improved half-life and glucose disposal in rodents. Ac-VPAC2P-PEG has therapeutic potential for diabetes management.

Intestinally Targeted Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitors Robustly Suppress Postprandial Triglycerides.

High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented.

Design of a long acting peptide functioning as both a glucagon-like peptide-1 receptor agonist and a glucagon receptor antagonist.

The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.

Contribution of site-specific PEGylation to the dipeptidyl peptidase IV stability of glucose-dependent insulinotropic polypeptide.

The effects of PEGylation of glucose-dependent insulinotropic polypeptide (GIP) on potency and dipeptidyl peptidase IV (DPPIV) stability are reported. N-terminal modification of GIP(1-30) with 40 kDa polyethylene glycol (PEG) abrogates functional activity. In contrast, C-terminal PEGylation of GIP(1-30) maintains full agonism and reasonable potency at the GIP receptor and confers a high level of DPPIV resistance. Moreover, the dual modification of N-terminal palmitoylation and C-terminal PEGylation results in a full agonist of comparable potency to native GIP that is stable to DPPIV cleavage. The results provide the basis for the development of long acting, PEGylated GIP, GIP variants, or GIP-based hybrid peptide therapeutics.

Specific inhibition of hormone-sensitive lipase improves lipid profile while reducing plasma glucose.

Elevation of plasma free fatty acids has been linked with insulin resistance and diabetes. Inhibition of lipolysis may provide a mechanism to decrease plasma fatty acids, thereby improving insulin sensitivity. Hormone-sensitive lipase (HSL) is a critical enzyme involved in the hormonally regulated release of fatty acids and glycerol from adipocyte lipid stores, and its inhibition may thus improve insulin sensitivity and blood glucose handling in type 2 diabetes. In rat adipocytes, forskolin-activated lipolysis was blocked by in vitro addition of a potent and selective HSL inhibitor or by prior treatment of the animals themselves. Antilipolytic effects also were demonstrated in overnight-fasted mice, rats, and dogs with species-dependent effects on plasma free fatty acid levels but with similar reductions in plasma glycerol being observed in all species. Inhibition of HSL also reduced hyperglycemia in streptozotocin-induced diabetic rats. The data support a connection between adipose tissue lipolysis and plasma glucose levels.

Mice deficient in the insulin-regulated membrane aminopeptidase show substantial decreases in glucose transporter GLUT4 levels but maintain normal glucose homeostasis.

The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.

In vitro SAR of (5-(2H)-isoxazolonyl) ureas, potent inhibitors of hormone-sensitive lipase.

A series of (5-(2H)-isoxazolonyl) ureas were developed as nanomolar inhibitors of hormone-sensitive lipase, an enzyme of potential importance in the treatment of diabetes.