Haim Tsubery

Reversible PEGylation of peptide YY3‐36 prolongs its inhibition of food intake in mice

Administration of peptide YY3‐36 (PYY3‐36) to fasting humans or mice shortly before re‐feeding effectively reduced their food intake, but PYY3‐36 exhibited a functional half‐life of only ∼3 h. Attachment of poly(ethylene glycol) to proteins and peptides (PEGylation) prolongs their half‐life in vivo, but completely inactivated PYY3‐36. We developed a reversibly PEGylated PYY3‐36 derivative by coupling it to a 40 kDa PEG through a spontaneously cleavable linker. The resulting conjugate (PEG40–FMS–PYY3‐36) gradually released unmodified PYY3‐36 in vivo, exhibiting an eightfold increase in its functional half‐life, to ∼24 h. This long‐acting PYY3‐36 pro‐drug may serve as an effective means for controlling food intake in humans.

N-terminal modifications of Polymyxin B nonapeptide and their effect on antibacterial activity.

Polymyxin B (PMB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. Polymyxin B nonapeptide (PMBN), the deacylated amino derivative of polymyxin B, is much less bactericidal but able to permeabilize the outer membrane of Gram-negative bacteria and to neutralize the toxic effects of lipopolysaccharide (LPS). In this study, we synthesized and evaluated the antibacterial and LPS neutralizing activities of four PMBN analogs modified at their N-terminal. Our results suggest that oligoalanyl substitutions of PMBN do not effect most of PMBN activities. However, a hydrophobic aromatic substitution generated a PMB-like molecule with high antibacterial activity and significant reduced toxicity.

Lipid binding and membrane penetration of polymyxin B derivatives studied in a biomimetic vesicle system

Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing D-instead of L-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane.

A Bacteriophage Capsid Protein Provides a General Amyloid Interaction Motif (GAIM) That Binds and Remodels Misfolded Protein Assemblies

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

[2-Sulfo-9-fluorenylmethoxycarbonyl]3-exendin-4: a long-acting glucose-lowering prodrug

Exendin-4, a glucagon-like peptide-1 agonist, is a relatively short-lived species in the circulatory system in vivo. We have linked three moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the three amino functions of exendin-4. FMS(3)-exendin-4 thus obtained has about 0.1% the glucose-lowering potency of the native peptide. Upon in vitro incubation at physiological conditions, the FMS moieties undergo hydrolysis generating the parent fully active, exendin-4. A single subcutaneous administration of FMS(3)-exendin-4 to healthy and type II diabetic mice has induced a glucose-lowering profile that exceeds in length several times that obtained by the native peptide. The reduction of blood glucose level began 1h after administration and was maximal 3-4h later. The blood glucose level returned to pre-administered values with t(1/2) of 12+/-0.7, 26+/-2, and 44+/-3h with doses of 1, 10, and 100 micro g/mouse, respectively. In sum, we have synthesized and characterized FMS(3)-exendin-4, a prodrug derivative of the native insulinotropic peptide, and found it to facilitate a prolonged and stable, glucose-lowering action in healthy and diabetic mice.

Suspensions of pro-drug insulin greatly prolong normoglycemic patterns in diabetic rats.

FMS(3)-insulin (2-sulfo-9-fluorenylmethoxycarbonyl)(3)-insulin is a water soluble, inactive-reactivated derivative of insulin with protracted action in vivo. In this study we find that FMS(3)-insulin preserves insulins capacity to crystallize when associated with Zn(2+) ions or with basic protamine. Zinc or protamine suspended preparations of FMS(3)-insulin manifest substantially prolonged, blood glucose-lowering pharmacokinetic profiles in STZ-treated rats (STZ-rats). A dose of up to 1mg suspended FMS(3)-insulin/STZ rat can be subcutaneously administered with no hypoglycemic episodes at any time point after administration. This dose yielded glucose-lowering profiles with t(1/2) values at the range of 50-70h, turning catabolic STZ-rats into anabolic ones over a period of 2-3 days. The obtained glucose-lowering patterns exceeded 7-8 times in duration those produced by nonhypoglycemic doses of NPH-insulin. In summary, subcutaneous administration of suspended insulin prodrugs, such as FMS(3)-insulin, can bring about prolonged, nonhypoglycemic glucose-lowering profiles, unattainable with insulin preparations, which are known to be active at the time of administration.

Conformation as the Therapeutic Target for Neurodegenerative Diseases.

Therapeutic strategies that target pathways of protein misfolding and the toxicity of intermediates along these pathways are mainly at discovery and early development stages, with the exception of monoclonal antibodies that have mainly failed to produce convincing clinical benefits in late stage trials. The clinical failures represent potentially critical lessons for future neurodegenerative disease drug development. More effective drugs may be achieved by pursuing the following two strategies. First, conformational targeting of aggregates of misfolded proteins, rather than less specific binding that includes monomer subunits, which vastly outnumber the toxic targets. Second, since neurodegenerative diseases frequently include more than one potential protein pathology, generic targeting of aggregates by shape might also be a crucial feature of a drug candidate. Incorporating both of these critical features into a viable drug candidate along with high affinity binding has not been achieved with small molecule approaches or with antibody fragments. Monoclonal antibodies developed so far are not broadly acting through conformational recognition. Using GAIM (General Amyloid Interaction Motif) represents a novel approach that incorporates high affinity conformational recognition for multiple protein assemblies, as well as recognition of an array of assemblies along the misfolding pathway between oligomers and fibers. A GAIM-Ig fusion, NPT088, is nearing clinical testing.

Oral therapy of L-glutamic acid γ-monohydroxamate-vanadium (2:1) complex: Improvement of blood glucose profile in different types of diabetic rodents

We report that oral administration of vanadium (+5) combined with L-glutamic acid γ-monohydroxamate at 1:2 stoichiometry [L-Glu(γ)HXM.VO3-] is highly effective in reducing blood glucose levels (BGLs) in a wide variety of diabetic rodents. In streptozocin-treated rats, a single administration (0.28 mmol/kg body wt) decreased BGL from 490 to 360 mg/dl within 1 h of administration, keeping this reduced level for additional 22 h, and a daily dose of 0.14 mmol/kg was found optimal. In Zucker diabetic fatty (ZDF) rats, a single dose of 0.14 mmol/kg normalized BGL within 8 h of administration, and maintained normal value for additional two days. In db/db mice, a single L-Glu(γ)HXM.VO3- administration of 0.2 mmol/kg decreased BGL from 500 ± 50 to 240 ± 20 mg/dl at 2 h, but was less effective afterwards. In high-carbohydrate (CHO)-fed Psammomis obesus, a single oral dose (0.14 mmol/kg) normalized BGL over a period of two days, and a daily dose of 0.07 mmol/kg/d, at the time P. obesus was transferred from low- to high-CHO diet, fully arrested the development of hyperglycemia characterizing this diabetic rodent. Finally, we found that the index of toxicity of orally administered L-GLU(γ)HXM-vanadate in rodents is 5-7 times lower than that of free sodium vanadate.

NPT088 reduces both amyloid-β and tau pathologies in transgenic mice

Alzheimers disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid‐β (Aβ) and hyper‐phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein (“NPT088”) consisting of the active fragment of g3p and human‐IgG1‐Fc.

Polymyxin B and Related Cyclic Peptides Facilitate Leanness and Reduce Fat Mass and Triglyceride Content in Ageing Rats: Potential Prototype Drugs Against Obesity

Polymyxin B (PMXB) blocks the action of insulin on glucose uptake in vitro. In vivo, it reverses hypoglycemia induced by exogenous insulin. Here we have treated mature male rats daily with PMXB over a period of two weeks. This therapy has decreased body weight by 11%, adipose fat mass by 46% and triglyceride levels by 39%, with no indication of liver or kidney toxicity. Two suboptimal parameters, however, were a decrease in food intake in the first week of treatment and some increase in fasting glucose levels. We have screened for PMXB-analogs having less associating affinity with rat-muscle phospholipids, and revealed that the same therapy using PMXB-derived peptide (nona-PMXB) is most optimal. This PMXB-analog is devoid of antibacterial activity and is four times less toxic than PMXB. Nona-PMXB therapy lower by 10, 32, 35 and 6% body weight gain, fat mass, circulating triglycerides and fasting glucose levels, respectively, in spite of normal or even elevated food intake in nona-PMXB treated rats. In summary, we found that nona-PMXB therapy is capable if inducing leanness in mature rats, particularly at the expense of decreasing fat-mass in adipose tissue. By and large, we suggest that lowering the action of insulin, on fat build-up solely, may be a therapeutically feasible task to fight with human adiposity in the future.