Hepatocyte Growth Factor and Macrophage-stimulating Protein "Hinge" Analogs to Treat Pancreatic Cancer.

Pancreatic cancer (PC) ranks twelfth in frequency of diagnosis but is the fourth leading cause of cancer related deaths with a 5 year survival rate of less than 7 percent. This poor prognosis occurs because the early stages of PC are often asymptomatic. Over-expression of several growth factors, most notably vascular endothelial growth factor (VEGF), has been implicated in PC resulting in dysfunctional signal transduction pathways and the facilitation of tumor growth, invasion and metastasis. Hepatocyte growth factor (HGF) acts via the Met receptor and has also received research attention with ongoing efforts to develop treatments to block the Met receptor and its signal transduction pathways. Macrophage-stimulating protein (MSP), and its receptor Ron, is also recognized as important in the etiology of PC but is less well studied. Although the angiotensin II (AngII)/AT1 receptor system is best known for mediating blood pressure and body water/electrolyte balance, it also facilitates tumor vascularization and growth by stimulating the expression of VEGF. A metabolite of AngII, angiotensin IV (AngIV) has sequence homology with the "hinge regions" of HGF and MSP, key structures in the growth factor dimerization processes necessary for Met and Ron receptor activation. We have developed AngIV-based analogs designed to block dimerization of HGF and MSP and thus receptor activation. Norleual has shown promise as tested utilizing PC cell cultures. Results indicate that cell migration, invasion, and pro-survival functions were suppressed by this analog and tumor growth was significantly inhibited in an orthotopic PC mouse model.

Contributions by the Brain Renin-Angiotensin System to Memory, Cognition, and Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive neuron losses in memory-associated brain structures that rob patients of their dignity and quality of life. Five drugs have been approved by the FDA to treat AD but none modify or significantly slow disease progression. New therapies are needed to delay the course of this disease with the ultimate goal of preventing neuron losses and preserving memory functioning. In this review we describe the renin-angiotensin II (AngII) system (RAS) with specific regard to its deleterious contributions to hypertension, facilitation of neuroinflammation and oxidative stress, reduced cerebral blood flow, tissue remodeling, and disruption of memory consolidation and retrieval. There is evidence that components of the RAS, AngIV and Ang(1-7), are positioned to counter such damaging influences and these systems are detailed with the goal of drawing attention to their importance as drug development targets. Ang(1-7) binds at the Mas receptor, while AngIV binds at the AT4 receptor subtype, and these receptor numbers are significantly decreased in AD patients, accompanied by declines in brain aminopeptidases A and N, enzymes essential for the synthesis of AngIV. Potent analogs may be useful to counter these changes and facilitate neuronal functioning and reduce apoptosis in memory associated brain structures of AD patients.

The Brain Hepatocyte Growth Factor/c-Met Receptor System: A New Target for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease increasing in frequency as life expectancy of the world's population increases. There are an estimated 5 million diagnosed AD patients in the U.S. and 16 million worldwide with no adequate treatment presently available. New therapeutic approaches are needed to slow, and hopefully reverse, disease progression. This review summarizes available information regarding an overlooked therapeutic target that may offer a treatment to slow and hopefully halt AD, namely the hepatocyte growth factor (HGF)/c-Met receptor system. Activation of the c-Met receptor stimulates mitogenesis, motogenesis, morphogenesis, the ability to mediate stem cell differentiation and neurogenesis, and protects against tissue insults in a wide range of cells including neurons. This growth factor system has recently been shown to induce dendritic arborization and synaptogenesis when stimulated by a newly developed angiotensin-based analogue, N-hexanoic-Tyr-Ile-(6) amino hexanoic amide (Dihexa). This small molecule was derived from the pre-prototype molecule Nle1-angiotensin IV and has shown promise in facilitating the formation of new functional synaptic connections and augmenting memory consolidation in animal models of AD. Dihexa is a first-in-class compound that is orally active, penetrates the blood-brain barrier, and facilitates memory consolidation and retrieval. This angiotensin-based small molecule may be efficacious as a treatment for AD.

The development of small molecule angiotensin IV analogs to treat Alzheimer's and Parkinson's diseases.

Alzheimer's (AD) and Parkinson's (PD) diseases are neurodegenerative diseases presently without effective drug treatments. AD is characterized by general cognitive impairment, difficulties with memory consolidation and retrieval, and with advanced stages episodes of agitation and anger. AD is increasing in frequency as life expectancy increases. Present FDA approved medications do little to slow disease progression and none address the underlying progressive loss of synaptic connections and neurons. New drug design approaches are needed beyond cholinesterase inhibitors and N-methyl-d-aspartate receptor antagonists. Patients with PD experience the symptomatic triad of bradykinesis, tremor-at-rest, and rigidity with the possibility of additional non-motor symptoms including sleep disturbances, depression, dementia, and autonomic nervous system failure. This review summarizes available information regarding the role of the brain renin-angiotensin system (RAS) in learning and memory and motor functions, with particular emphasis on research results suggesting a link between angiotensin IV (AngIV) interacting with the AT4 receptor subtype. Currently there is controversy over the identity of this AT4 receptor protein. Albiston and colleagues have offered convincing evidence that it is the insulin-regulated aminopeptidase (IRAP). Recently members of our laboratory have presented evidence that the brain AngIV/AT4 receptor system coincides with the brain hepatocyte growth factor/c-Met receptor system. In an effort to resolve this issue we have synthesized a number of small molecule AngIV-based compounds that are metabolically stable, penetrate the blood-brain barrier, and facilitate compromised memory and motor systems. These research efforts are described along with details concerning a recently synthesized molecule, Dihexa that shows promise in overcoming memory and motor dysfunctions by augmenting synaptic connectivity via the formation of new functional synapses.

The procognitive and synaptogenic effects of angiotensin IV-derived peptides are dependent on activation of the hepatocyte growth factor/c-met system.

A subset of angiotensin IV (AngIV)-related molecules are known to possess procognitive/antidementia properties and have been considered as templates for potential therapeutics. However, this potential has not been realized because of two factors: 1) a lack of blood-brain barrier-penetrant analogs, and 2) the absence of a validated mechanism of action. The pharmacokinetic barrier has recently been overcome with the synthesis of the orally active, blood-brain barrier-permeable analog N-hexanoic-tyrosine-isoleucine-(6) aminohexanoic amide (dihexa). Therefore, the goal of this study was to elucidate the mechanism that underlies dihexa's procognitive activity. Here, we demonstrate that dihexa binds with high affinity to hepatocyte growth factor (HGF) and both dihexa and its parent compound Norleucine 1-AngIV (Nle(1)-AngIV) induce c-Met phosphorylation in the presence of subthreshold concentrations of HGF and augment HGF-dependent cell scattering. Further, dihexa and Nle(1)-AngIV induce hippocampal spinogenesis and synaptogenesis similar to HGF itself. These actions were inhibited by an HGF antagonist and a short hairpin RNA directed at c-Met. Most importantly, the procognitive/antidementia capacity of orally delivered dihexa was blocked by an HGF antagonist delivered intracerebroventricularly as measured using the Morris water maze task of spatial learning.

A Role for the Brain RAS in Alzheimer's and Parkinson's Diseases.

The brain renin-angiotensin system (RAS) has available the necessary functional components to produce the active ligands angiotensins II (AngII), angiotensin III, angiotensins (IV), angiotensin (1-7), and angiotensin (3-7). These ligands interact with several receptor proteins including AT1, AT2, AT4, and Mas distributed within the central and peripheral nervous systems as well as local RASs in several organs. This review first describes the enzymatic pathways in place to synthesize these ligands and the binding characteristics of these angiotensin receptor subtypes. We next discuss current hypotheses to explain the disorders of Alzheimer's disease (AD) and Parkinson's disease (PD), as well as research efforts focused on the use of angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs), in their treatment. ACE inhibitors and ARBs are showing promise in the treatment of several neurodegenerative pathologies; however, there is a need for the development of analogs capable of penetrating the blood-brain barrier and acting as agonists or antagonists at these receptor sites. AngII and AngIV have been shown to play opposing roles regarding memory acquisition and consolidation in animal models. We discuss the development of efficacious AngIV analogs in the treatment of animal models of AD and PD. These AngIV analogs act via the AT4 receptor subtype which may coincide with the hepatocyte growth factor/c-Met receptor system. Finally, future research directions are described concerning new approaches to the treatment of these two neurological diseases.

ST6Gal-I sialyltransferase confers cisplatin resistance in ovarian tumor cells.

BACKGROUND: Platinum drugs, including cisplatin, are a frontline therapeutic in ovarian cancer treatment and acquired resistance to these agents is a major contributor to ovarian cancer morbidity and mortality. In this study a novel glycosylation-dependent mechanism for cisplatin resistance is described. Specifically, cisplatin-induced cell death is blocked by the activity of the ST6Gal-I sialyltransferase. ST6Gal-I modifies specific receptors by adding a negatively charged sialic acid sugar which influences diverse receptor functions. Overexpression of ST6Gal-I is a hallmark of ovarian and other cancers and its expression has been correlated to metastasis and poor prognosis. METHODS: Tumor cell viability and apoptotic induction were determined in cell lines with ST6Gal-I overexpression and knockdown. In addition, cell populations with acquired resistance to cisplatin were assayed for endogenous ST6Gal-I expression. RESULTS: We show that forced expression of ST6Gal-I in OV4 ovarian cancer cells that lack endogenous ST6Gal-I causes reduced activation of caspase 3 and increased cell viability following cisplatin treatment. Conversely, forced ST6Gal-I knockdown in Pa-1 cells with high endogenous ST6Gal-I increases cisplatin-induced caspase activation and cell death. A2780 ovarian cancer cells selected for stable cisplatin resistance display upregulated endogenous ST6Gal-I when compared with parental, cisplatin-sensitive, A2780 cells. Similarly, extended low dose cisplatin treatment of a Pa-1 polyclonal ST6Gal-I shRNA knockdown population led to selection for subclones with elevated ST6Gal-I expression. CONCLUSIONS: Receptor sialylation by ST6Gal-I confers a survival advantage for tumor cells in the presence of cisplatin. These collective findings support a role for ST6Gal-I in chemoresistance and highlight ST6Gal-I as a potential therapeutic target for platinum resistant tumors.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin.

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 µM) and PD123319 (10 µM) plus 100 µM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1-20 µM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 µM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

The brain renin-angiotensin system: a diversity of functions and implications for CNS diseases.

The classic renin-angiotensin system (RAS) was initially described as a hormone system designed to mediate cardiovascular and body water regulation, with angiotensin II as its major effector. The discovery of an independent local brain RAS composed of the necessary functional components (angiotensinogen, peptidases, angiotensins, and specific receptor proteins) significantly expanded the possible physiological and pharmacological functions of this system. This review first describes the enzymatic pathways resulting in active angiotensin ligands and their interaction with AT(1), AT(2), and AT(4) receptor subtypes. Next, we discuss the classic physiologies and behaviors controlled by the RAS including cardiovascular, thirst, and sodium appetite. A final section summarizes non-classic functions and clinical conditions mediated by the brain RAS with focus on memory and Alzheimer's disease. There is no doubt that the brain RAS is an important component in the development of dementia. It also appears to play a role in normal memory consolidation and retrieval. The presently available anti-dementia drugs are proving to be reasonably ineffective, thus alternative treatment approaches must be developed. At the same time, presently available drugs must be tested for their efficacy to treat newly identified syndromes and diseases connected with the RAS. The list of non-classic physiologies and behaviors is ever increasing in both number and scope, attesting to the multidimensional influences of the RAS. Such diversity in function presents a dilemma for both researchers and clinicians. Namely, the blunting of RAS subsystems in the hopes of combating one constellation of underlying causes and disease symptoms may be counter-balanced by unanticipated and unwanted consequences to another RAS subsystem. For example, the use of angiotensin-converting enzyme inhibitors and AT(1) and/or AT(2) receptor blockers have shown great promise in the treatment of cardiovascular related pathologies; however, their use could negate the cerebroprotective benefits offered by this system.

Evaluation of metabolically stabilized angiotensin IV analogs as procognitive/antidementia agents.

Angiotensin IV (AngIV: VYIHPF)-related peptides have long been recognized as procognitive agents with potential as antidementia therapeutics. Their development as useful therapeutics, however, has been limited by physiochemical properties that make them susceptible to metabolic degradation and impermeable to gut and blood-brain barriers. A previous study demonstrated that the core structural information required to impart the procognitive activity of the AngIV analog, norleucine(1)-angiotensin IV, resides in its three N-terminal amino acids, Nle-Tyr-Ile. The goal of this project was to chemically modify this tripeptide in such a way to enhance its metabolic stability and barrier permeability to produce a drug candidate with potential clinical utility. Initial results demonstrated that several N- and C-terminal modifications lead to dramatically improved stability while maintaining the capability to reverse scopolamine-induced deficits in Morris water maze performance and augment hippocampal synaptogenesis. Subsequent chemical modifications, which were designed to increase hydrophobicity and decrease hydrogen bonding, yielded an orally active, blood-barrier permeant, metabolically stabilized analog, N-hexanoic-Tyr-Ile-(6) aminohexanoic amide (dihexa), that exhibits excellent antidementia activity in the scopolamine and aged rat models and marked synaptogenic activity. These data suggest that dihexa may have therapeutic potential as a treatment of disorders, such as Alzheimer's disease, where augmented synaptic connectivity may be beneficial.

A role for matrix metalloproteinases in nicotine-induced conditioned place preference and relapse in adolescent female rats.

Reconfiguration of extracellular matrix proteins appears to be necessary for the synaptic plasticity that underlies memory consolidation. The primary candidates involved in controlling this process are a family of endopeptidases called matrix metalloproteinases (MMPs); however, the potential role of MMPs in nicotine addiction-related memories has not been adequately tested. Present results indicate transient changes in hippocampal MMP-2, -3, and -9 expression following context dependent learning of nicotine-induced conditioned place preference (CPP). Members of a CPP procedural control group also indicated similar MMP changes, suggesting that memory activation occurred in these animals as well. However, hippocampal MMP-9 expression was differentially elevated in members of the nicotine-induced CPP group on days 4 and 5 of training. Inhibition of MMPs using a broad spectrum MMP inhibitor (FN439) during nicotine-induced CPP training blocked the acquisition of CPP. Elevations in hippocampal and prefrontal cortex MMP-3 expression-but not MMP-2 and -9-accompanied reactivation of a previously learned drug related memory. Decreases in the actin regulatory cytoskeletal protein cortactin were measured in the HIP and PFC during the initial two days of acquisition of CPP; however, no changes were seen following re-exposure to the drug related environment. These results suggest that MMP-9 may be involved in facilitating the intracellular and extracellular events required for the synaptic plasticity underlying the acquisition of nicotine-induced CPP. Furthermore, MMP-3 appears to be important during re-exposure to the drug associated environment. However, rats introduced into the CPP apparatus and given injections of vehicle rather than nicotine during training also revealed a pattern of MMP expression similar to nicotine-induced CPP animals.

Importance of the brain Angiotensin system in Parkinson's disease.

Parkinson's disease (PD) has become a major health problem affecting 1.5% of the world's population over 65 years of age. As life expectancy has increased so has the occurrence of PD. The primary direct consequence of this disease is the loss of dopaminergic (DA) neurons in the substantia nigra and striatum. As the intensity of motor dysfunction increases, the symptomatic triad of bradykinesia, tremors-at-rest, and rigidity occur. Progressive neurodegeneration may also impact non-DA neurotransmitter systems including cholinergic, noradrenergic, and serotonergic, often leading to the development of depression, sleep disturbances, dementia, and autonomic nervous system failure. L-DOPA is the most efficacious oral delivery treatment for controlling motor symptoms; however, this approach is ineffective regarding nonmotor symptoms. New treatment strategies are needed designed to provide neuroprotection and encourage neurogenesis and synaptogenesis to slow or reverse this disease process. The hepatocyte growth factor (HGF)/c-Met receptor system is a member of the growth factor family and has been shown to protect against degeneration of DA neurons in animal models. Recently, small angiotensin-based blood-brain barrier penetrant mimetics have been developed that activate this HGF/c-Met system. These compounds may offer a new and novel approach to the treatment of Parkinson's disease.

Focus on Brain Angiotensin III and Aminopeptidase A in the Control of Hypertension.

The classic renin-angiotensin system (RAS) was initially described as a hormone system designed to mediate cardiovascular and body water regulation. The discovery of a brain RAS composed of the necessary functional components (angiotensinogen, peptidases, angiotensins, and specific receptor proteins) independent of the peripheral system significantly expanded the possible physiological and pharmacological functions of this system. This paper first describes the enzymatic pathways resulting in active angiotensin ligands and their interaction with AT(1), AT(2), and mas receptor subtypes. Recent evidence points to important contributions by brain angiotensin III (AngIII) and aminopeptidases A (APA) and N (APN) in sustaining hypertension. Next, we discuss current approaches to the treatment of hypertension followed by novel strategies that focus on limiting the binding of AngII and AngIII to the AT(1) receptor subtype by influencing the activity of APA and APN. We conclude with thoughts concerning future treatment approaches to controlling hypertension and hypotension.

Insulin-sensitizing effects on muscle and adipose tissue after dietary fiber intake in men and women with metabolic syndrome.

CONTEXT: Dietary fibers have been associated with a reduced incidence of type 2 diabetes mellitus in epidemiological studies; however, the precise mechanisms are unknown. OBJECTIVE: The objective of the study was to evaluate the efficacy and site of action of an insoluble dietary fiber derived from maize (HAM-RS2) in improving insulin resistance in subjects at increased risk of type 2 diabetes mellitus. DESIGN: This study was a randomized, controlled crossover, dietary intervention study. SETTING: The study was conducted at the Centre for Diabetes, Endocrinology, and Research, Royal Surrey County Hospital, Guildford, United Kingdom. PARTICIPANTS: Fifteen men and women with insulin resistance participated in the study. INTERVENTION: The intervention included 40 g/d HAM-RS2 compared with a matched placebo for 8 wk. MAIN OUTCOME MEASURES: After each supplement, participants underwent a two-step hyperinsulinemic-euglycemic clamp study with the addition of glucose tracers; a meal tolerance test; arteriovenous sampling across forearm muscle tissue; and a sc adipose tissue biopsy for assessment of gene expression. RESULTS: There was enhanced uptake of glucose into the forearm muscle measured by arteriovenous sampling (65 ± 15% increase after resistant starch; P < 0.001). Adipose tissue function was also affected, with enhanced fatty acid suppression after HAM-RS2 treatment and an increase in gene expression for hormone sensitive lipase (P = 0.005), perilipin (P = 0.011), lipoprotein lipase (P = 0.014), and adipose triglyceride lipase (P = 0.03) in biopsy samples. There was no effect on the insulin sensitivity of hepatic glucose production or plasma lipids after HAM-RS2. CONCLUSION: HAM-RS2 improved peripheral but not hepatic insulin resistance and requires further study as an intervention in patients with or at risk for type 2 diabetes.

A Period 2 genetic variant interacts with plasma SFA to modify plasma lipid concentrations in adults with metabolic syndrome.

Genetic variants of Period 2 (PER2), a circadian clock gene, have been linked to metabolic syndrome (MetS). However, it is still unknown whether these genetic variants interact with the various types of plasma fatty acids. This study investigated whether common single nucleotide polymorphisms (SNPs) in the PER2 locus (rs934945 and rs2304672) interact with various classes of plasma fatty acids to modulate plasma lipid metabolism in 381 participants with MetS in the European LIPGENE study. Interestingly, the rs2304672 SNP interacted with plasma total SFA concentrations to affect fasting plasma TG, TG-rich lipoprotein (TRL-TG), total cholesterol, apoC-II, apoB, and apoB-48 concentrations (P-interaction < 0.001-0.046). Carriers of the minor allele (GC+GG) with the highest SFA concentration (>median) had a higher plasma TG concentration (P = 0.001) and higher TRL-TG (P < 0.001) than the CC genotype. In addition, participants carrying the minor G allele for rs2304672 SNP and with a higher SFA concentration (>median) had higher plasma concentrations of apo C-II (P < 0.001), apo C-III (P = 0.009), and apoB-48 (P = 0.028) compared with the homozygotes for the major allele (CC). In summary, the rs2304672 polymorphism in the PER2 gene locus may influence lipid metabolism by interacting with the plasma total SFA concentration in participants with MetS. The understanding of these gene-nutrient interactions could help to provide a better knowledge of the pathogenesis in MetS.

The Hepatocyte Growth Factor/c-Met Antagonist, Divalinal-Angiotensin IV, Blocks the Acquisition of Methamphetamine Dependent Conditioned Place Preference in Rats.

The use of methamphetamine (MA) is increasing in the U.S. and elsewhere around the world. MA's capacity to cause addiction significantly exceeds other psychostimulant drugs, and its use negatively impacts learning and memory. Recently, attempts have been made to interfere with the presumed mechanism(s) underlying the establishment of drug-induced memory consolidation. The majority of these studies have employed matrix metalloproteinase (MMP) inhibitors to disrupt MMP-induced extracellular matrix molecule dependent synaptic reconfiguration, or GABA receptor agonists. The present investigation utilized an angiotensin IV (AngIV) analogue, Divalinal-AngIV (divalinal), to disrupt acquisition of MA-induced dependence in rats as measured using the conditioned place preference paradigm. Results indicate that both acute and chronic intracerebroventricular infusion of divalinal prior to each daily subcutaneous injection of MA prevented acquisition. However, divalinal was unable to prevent MA-induced reinstatement after prior acquisition followed by extinction trials. These results indicate that prevention of MA dependence can be accomplished by blockade of the brain AT4 receptor subtype. On the other hand, once MA-induced memory consolidation is in place divalinal appears to be ineffective. Mechanistic studies indicated that divalinal is a potent inhibitor of the hepatocyte growth factor (HGF)/c-Met receptor system, and thus it appears that a functional HGF/c-Met system is required for the acquisition of MA-mediated conditioned place preference.

Development of angiotensin IV analogs as hepatocyte growth factor/Met modifiers.

The 6-AH family [D-Nle-X-Ile-NH-(CH(2))(5)-CONH(2); where X = various amino acids] of angiotensin IV (Ang IV) analogs binds directly to hepatocyte growth factor (HGF) and inhibit HGF's ability to form functional dimers. The metabolically stabilized 6-AH family member, D-Nle-Tyr-Ile-NH-(CH(2))(5)-CONH(2,) had a t(1/2) in blood of 80 min compared with the parent compound norleual [Nle-Tyr-Leu-Ψ-(CH(2)-NH(2))(3-4)-His-Pro-Phe], which had a t(1/2) in blood of <5 min. 6-AH family members were found to act as mimics of the dimerization domain of HGF (hinge region) and inhibited the interaction of an HGF molecule with a (3)H-hinge region peptide resulting in an attenuated capacity of HGF to activate its receptor Met. This interference translated into inhibition of HGF-dependent signaling, proliferation, and scattering in multiple cell types at concentrations down into the low picomolar range. We also noted a significant correlation between the ability of the 6-AH family members to block HGF dimerization and inhibition of the cellular activity. Furthermore, a member of the 6-AH family with cysteine at position 2, was a particularly effective antagonist of HGF-dependent cellular activities. This compound suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system. Together, these data indicate that the 6-AH family of Ang IV analogs exerts its biological activity by modifying the activity of the HGF/Met system and offers the potential as therapeutic agents in disorders that are dependent on or possess an overactivation of the HGF/Met system.

Mimics of the dimerization domain of hepatocyte growth factor exhibit anti-Met and anticancer activity.

The angiotensin IV analog norleual [Nle-Tyr-Leu-ψ-(CH(2)-NH(2))-Leu-His-Pro-Phe] has been shown recently to act as a hepatocyte growth factor (HGF)/Met antagonist capable of blocking the binding of HGF to the Met receptor, inhibiting HGF-dependent activation of Met, and attenuating HGF-dependent cellular activities. In addition, norleual exhibited marked anticancer activity. Homology between norleual and the dimerization domain (hinge region) of HGF led to the hypothesis that norleual acts by interfering with HGF dimerization/multimerization and functions as a dominant-negative hinge region mimic. To test this hypothesis we investigated the ability of norleual to bind to and inhibit the dimerization of HGF. To further evaluate the idea that norleual was acting as a hinge region mimic, we synthesized a hexapeptide representing the HGF hinge sequence and established its capacity to similarly block HGF-dependent activation of Met and HGF-dependent cellular functions. The hinge peptide not only bound with high affinity directly to HGF and blocked its dimerization but it also inhibited HGF-dependent Met activation, suppressed HGF-dependent cellular functions, and exhibited anticancer activity. The major implication of this study is that molecules targeting the dimerization domain of HGF may represent novel and viable anticancer therapeutic agents; the development of such molecules should be feasible using norleual and the hinge peptide as synthetic templates.

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation.

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.

Facilitation of hippocampal synaptogenesis and spatial memory by C-terminal truncated Nle1-angiotensin IV analogs.

Angiotensin IV (AngIV; Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6))-related peptides have emerged as potential antidementia agents. However, their development as practical therapeutic agents has been impeded by a combination of metabolic instability, poor blood-brain barrier permeability, and an incomplete understanding of their mechanism of action. This study establishes the core structure contained within norleucine(1)-angiotensin IV (Nle(1)-AngIV) that is required for its procognitive activity. Results indicated that Nle(1)-AngIV-derived peptides as small as tetra- and tripeptides are capable of reversing scopolamine-induced deficits in Morris water maze performance. This identification of the active core structure contained within Nle(1)-AngIV represents an initial step in the development of AngIV-based procognitive drugs. The second objective of the study was to clarify the general mechanism of action of these peptides by assessing their ability to affect changes in dendritic spines. A correlation was observed between a peptide's procognitive activity and its capacity to increase spine numbers and enlarge spine head size. These data suggest that the procognitive activity of these molecules is attributable to their ability to augment synaptic connectivity.