Search

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The blood-brain barrier and the blood-cerebrospinal fluid barrier prevent access of biotherapeutics to their targets in the central nervous system and therefore prohibit the effective treatment of neurological disorders. In an attempt to discover novel brain transport vectors in vivo, we injected a T7 phage peptide library and continuously collected blood and cerebrospinal fluid (CSF) using a cisterna magna cannulated conscious rat model. Specific phage clones were highly enriched in the CSF after four rounds of selection. Validation of individual peptide candidates showed CSF enrichments of greater than 1000-fold. The biological activity of peptide-mediated delivery to the brain was confirmed using a BACE1 peptide inhibitor linked to an identified novel transport peptide which led to a 40% reduction of Amyloid-β in CSF. These results indicate that the peptides identified by the in vivo phage selection approach could be useful transporters for systemically administrated large molecules into the brain with therapeutic benefits.

The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting., (Copyright © 2015. Published by Elsevier B.V.)

Melittin is an amphipathic peptide which has received much attention as a model peptide for peptide-membrane interactions. It is however not suited as a transfection agent due to its cytolytic and toxicological effects. Retro-inverso-melittin, when covalently linked to the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (riDOM), eliminates these shortcomings. The interaction of riDOM with phospholipid membranes was investigated with circular dichroism (CD) spectroscopy, dynamic light scattering, ζ-potential measurements, and high-sensitivity isothermal titration calorimetry. riDOM forms cationic nanoparticles with a diameter of ~13nm which are well soluble in water and bind with high affinity to DNA and lipid membranes. When dissolved in bilayer membranes, riDOM nanoparticles dissociate and form transient pores. riDOM-induced membrane leakiness is however much reduced compared to that of authentic melittin. The secondary structure of the ri-melittin is not changed when riDOM is transferred from water to the membrane and displays a large fraction of β-structure. The (31)P NMR spectrum of the nanoparticle is however transformed into a typical bilayer spectrum. The Gibbs free energy of riDOM binding to bilayer membranes is -8.0 to -10.0kcal/mol which corresponds to the partition energy of just one fatty acyl chain. Half of the hydrophobic surface of the riDOM lipid extension with its 2 oleic acyl chains is therefore involved in a lipid-peptide interaction. This packing arrangement guarantees a good solubility of riDOM both in the aqueous and in the membrane phase. The membrane binding enthalpy is small and riDOM binding is thus entropy-driven., (© 2013. Published by Elsevier B.V. All rights reserved.)

DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

A novel sulfonylureido pyridine series exemplified by compound 19 yielded potent inhibitors of FBPase showing significant glucose reduction and modest glycogen lowering in the acute db/db mouse model for Type-2 diabetes. Our inhibitors occupy the allosteric binding site and also extend into the dyad interface region of tetrameric FBPase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Sulfonylureido thiazoles were identified from a HTS campaign and optimized through a combination of structure-activity studies, X-ray crystallography and molecular modeling to yield potent inhibitors of fructose-1,6-bisphosphatase. Compound 12 showed favorable ADME properties, for example, F=70%, and a robust 32% glucose reduction in the acute db/db mouse model for Type-2 diabetes., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Human fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) is a key gluconeogenic enzyme, responsible for the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate, and thus presents an opportunity for the development of novel therapeutics focused on lowering the hepatic glucose production in type 2 diabetics. In its active form FBPase exists as a homotetramer and is allosterically regulated by AMP. In an HTS campaign aromatic sulfonylureas have been identified as FBPase inhibitors mimicking AMP. By bridging two adjacent allosteric binding sites using two aromatic sulfonylureas as anchor units and covalently linking them, it was possible to obtain dual binding AMP site inhibitors that exhibit a strong inhibitory effect.

Melittin is an amphipathic cationic peptide derived from honeybee venom with well-known cytolytic and antimicrobial properties. When coupled to cationic polymers or lipid molecules, it forms conjugates with high transfection efficiency and low toxicity with promising applications in gene therapy. A first step in the internalization of melittin and its conjugates is their binding to the cell surface, a reaction likely to involve heparan sulfate proteoglycans (HSPG). In the present work, we characterize the binding equilibrium of heparan sulfate (HS) with two melittin analogues, [Cys(1)]melittin (mel-SH) and retro-inverso [Cys(1)]melittin (ri-mel-SH). The terminal cysteine found in these peptides replaces the N-terminal glycine present in native melittin and allows covalent binding to other molecules. Isothermal titration calorimetry (ITC) reveals a high affinity of each melittin analogue to HS. Association constants of 4.7 x 10(4) and 3.5 x 10(5) M(-)(1) are found at physiological ionic strength and 15 degrees C for ri-mel-SH and mel-SH, respectively. The reaction enthalpy measured under these conditions is DeltaH(degrees)pep= 4.2 kcal/mol for ri-mel-SH and DeltaH(degrees)pep= 1.1 kcal/mol for mel-SH. The peptide-to-HS stoichiometry is approximately 20 for ri-mel-SH and approximately 14 for mel-SH under the same conditions. Temperature dependence studies using ri-mel-SH (mel-SH) show that DeltaH(degrees)pep decreases in magnitude upon increase in temperature, which results in a molar heat capacity of DeltaH(degrees)pep= -322 cal mol(-)(1) K(-)(1) (-45 cal mol(-)(1) K(-)(1)). Such a negative heat capacity change is not expected for a purely electrostatic interaction and indicates that hydrophobic and other interactions are also involved in the binding equilibrium. Salt dependence studies of the binding constants confirm that nonelectrostatic forces are an important component of the HS-melittin interaction. Binding to HS induces conformational changes in both peptides, with ri-mel-SH showing a 6-fold increase of the alpha-helix content when incubated with HS under saturation conditions.

Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting that binding takes place at some distance from the plane of the polar groups. (31)P NMR indicates that the lipid bilayer remains intact upon R(9) binding. The fact that R(9) binds with greater affinity to HS than to anionic lipid vesicles makes the former molecule a more likely target in binding this CPP to the cell surface.

Starting from the tetrapeptide Ac-pYEEI-NHMe and using a structure-based approach, we have designed and synthesised a peptidomimetic ligand for p56(lck) SH2 domain containing a conformationally restricted replacement for the two glutamate residues. We have explored replacments for the isoleucine residue in the pY+3 pocket and thus identified 1-(R)-amino-3-(S)-indaneacetic acid as the most potent replacement. We also report the X-ray crystal structures of two of the antagonists.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to approximately 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ-Lucifer yellow (DS3). The Km values obtained were 1.7 microM and 6.2 microM, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 degrees C.

In order to obtain experimental data on the question of compensation of positive charges by anionic groups in multiply charged ions of polyfunctional molecules in electrospray MS, several pairs of peptides with the same basic structure but differentiated by one or two strongly acidic groups (phosphate or sulfonate) were investigated. It was found that depending on the density of basic centers and the strength of acidic groups present, the observed changes upon introduction of acidic groups ranged from complete elimination of the most highly charged state to absence of any difference.

The synthon for O-thiophosphotyrosine, Fmoc-Tyr[PS(OBzl)2]-OH (1c), was prepared in 63% yield from Fmoc-Tyr-OH by first transient protection as the tBuMe2Si-ester and phosphinylation with (BzlO)2PNiPr2/tetrazole followed by oxidation of P(III) to P(V) with S8 in CS2. Building block 1c was incorporated in the Fmoc solid-phase synthesis of two O-thiophosphotyrosine-containing peptides H-Thr-Glu-Pro-Gln-Tyr(PS)-Gln-Pro-Gly-Glu-OH (2) and H-Thr-Arg-Asp-Ile-Tyr(PS)-Glu-Thr-Asp-Phe-Phe-Arg-Lys-OH (3), corresponding to sequences of the p60src (523-531) protein and an insulin receptor (IR) (1142-1153) analogue, respectively. An alternative approach of synthesis, the global phosphorylation of a resin-bound peptide, also proved useful. Thus, the free tyrosyl side-chain containing-peptide IR (1142-1153) on support was phosphinylated with the above phosphoramidite reagent followed by oxidation with either S8/CS2 or tetraethylthiuram disulfide/CH3CN solutions. Deprotection and peptide-resin cleavage was performed with a TFA/thiophenol (H2O) mixture. Crude peptides 2 and 3 were stable to the acidolytic deprotection. Preparative RP(C18)HPLC was initially performed using 0.1% TFA(aq)/EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosphopeptide in solution. Stability measurements both as a function of time and pH, further confirmed this initial finding. Purifications performed at intermediate pH using a triethylammonium acetate (pH 7.5)/CH3CN solvent system overcame this problem.

The derivative N alpha-fluorenylmethoxy-carbonyl-O-dimethylphosphono-L-tyrosi ne was utilized in continuous-flow solid-phase synthesis to prepare the O-phosphotyrosine analogue of human angiotensin II, [Tyr(P)4] angiotensin II. Side-chain deprotection, including the removal of the methyl phosphate groups, as well as cleavage of the peptide from the solid support was achieved with 1 M trimethylsilyl-bromide-thioanisole in trifluoroacetic acid. Overall yield of purified peptide was 46%. The pressor response in intact sheep to graded infusions of the synthetic [Tyr(P)4] angiotensin II showed it to have similar potency to the native angiotensin II. However, a prolonged, elevated mean arterial pressure was observed following cessation of the infusion.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.

An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142-1,153 of the insulin receptor was prepared and incubated with the 40-kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H-NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri-, di-, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.

A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.

The synthetic phosphotyrosyl tridecapeptide H-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-Gln-Gly-OH, reproducing a major phosphoacceptor site of protein tyrosine kinases of the src-family, can be phosphorylated at Thr-9 by both casein kinases -1 and -2. Its shorter derivative H-Asn-Glu-Tyr(P)-Thr-Ala-OH is not affected by casein kinase-1 while representing a substrate as good as the tridecapeptide for casein kinase-2. The unphosphorylated analogue H-Asn-Glu-Tyr-Thr-Ala-OH, however, is a much poorer substrate, and no significant phosphorylation could be observed of its O-methyl ether derivative H-Asn-Glu-Tyr(Me)-Thr-Ala-OMe. These data on one side corroborate the concept that casein kinase-1 recognizes residues located on the C-terminal edge of acidic stretches, providing, on the other, the evidence that phosphotyrosyl side chains can act as specificity determinants for casein kinase-2.