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Biomedical subjects

A M Felix

Publications and source records attributed to A M Felix.

At least 19 recordsLinked to original sources
PubMed2005

Synthesis of bis- and tris-branched COOH-terminal pegylating reagents: conjugation to NH-terminal peptides.

In previous studies we reported an orthogonal protection scheme that was developed for the solution-phase synthesis of a family of bis- and tris-pegylating reagents which contain a free NH(2)-terminus. These pegylating reagents were coupled to the COOH-terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis- and tris-pegylating reagents which contain a free COOH-terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH(2)-function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH- or NH(2)-functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.

Amines↗
PubMed2004

Synthesis of symmetrically and asymmetrically branched pegylating reagents.

A solution-phase procedure using an orthogonal protection scheme was developed for the synthesis of a novel family of multi-pegylating reagents. The procedure was exemplified by the synthesis of bis- and tris-pegylating reagents prepared by stepwise insertion of the poly(ethylene glycol) units thereby enabling the preparation of both symmetrical and asymmetrical pegylating reagents. Asymmetrical pegylation and tris-pegylation of peptides and proteins introduces new variables for use in the optimization of pegylated peptides and proteins. These reagents are ideally suited for conjugation to peptides and proteins as they possess a required functional group and will be useful intermediates for the synthesis of a new generation of pegylated products. Tris-pegylation can also provide more effective protection from proteolysis by shielding the protein surface from approaching macromolecules. To illustrate this potential, conditions were developed for the successful coupling of the tris-pegylating reagent to a model pentapeptide.

Indicators and Reagents↗
PubMed2000

Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid.

Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Alzheimer Disease↗
PubMed2000

The Alzheimer's peptide a beta adopts a collapsed coil structure in water.

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Amyloid beta-Peptides↗
PubMed1998

Combined solid-phase/solution synthesis of a 31-residue vasoactive intestinal peptide analog: general method for repetitive coupling of fragments without isolation and purification of intermediates.

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo-(Lys21-Asp25)Ac[Glu8 Lys12 Nle17 Ala19, Asp25 Leu26,Lys27,28,Gly29,30,Thr31]-VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other beta-agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium-sized hentriacontapeptide. From these studies we developed a combined solid-phase/solution synthesis which uses four protected fragments (each prepared by solid-phase synthesis with highly acid-labile resins) possessing Nalpha-9-fluorenylmethyloxycarbonyl and side-chain tert-butyl protection. Only equivalent amounts of each fragment were required to achieve near-quantitative coupling reactions using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmeth anaminium hexafluorophosphate N-oxide/N-hydroxybenzotriazole. All reagents and side products were removed at each stage by simple extraction procedures. Final deprotection was carried out with 90% trifluoroacetic acid. Under these conditions only low levels of epimerization were observed (<2%). These diastereoisomers and other trace impurities were removed from the product in a single purification by preparative high-performance liquid chromatography. The procedure has been scaled up (10-g scale) and the final product obtained in an overall (nonoptimized) yield of 24%. This procedure for the repetitive coupling of fragments, without isolation of intermediates, may be generally applicable for the economic synthesis of other medium-sized and longer peptides.

Amino Acid Sequence↗
PubMed1997

A beta deposition inhibitor screen using synthetic amyloid.

The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Amyloid↗
PubMed1997

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo.

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.

Animals↗
PubMed1996

Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence.

Alzheimer's disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Alzheimer Disease↗
PubMed1995

Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.

Adolescent↗
PubMed1995

1H NMR of A beta amyloid peptide congeners in water solution. Conformational changes correlate with plaque competence.

To begin to examine the structural basis for the deposition of soluble A beta amyloid peptide onto senile plaques in Alzheimer's disease, we have prepared A beta congeners and measured their activity in an in vitro plaque growth assay. The N-terminal fragment, A beta (1-28)-OH, was inactive at all pH values tested. While the central fragment, A beta (10-35)-NH2, and the full length peptide, A beta (1-40)-OH, were inactive below pH 4, both were active (plaque competent) between pH 5 and 9. The active and inactive fragments were studied by nuclear magnetic resonance spectroscopy in water at submillimolar concentrations at pH 2.1 and 5.6. Changes in chemical shifts, coupling constants, and nuclear Overhauser enhancements indicate a pH dependent folding transition in A beta (10-35)-NH2 as it becomes active. The conformation of the active fragment is not helical, and preliminary data indicate the presence of several turns and at least two short strands. In contrast, the inactive fragment A beta (1-28)-OH did not undergo a similar folding transition. Earlier nuclear magnetic resonance studies of amyloid peptides in fluorinated alcohols or detergent micelles at low pH described a helical conformation and proposed a helix to sheet transition in plaque formation; the present study demonstrates that no such conformations are present in water under conditions where the peptides can adhere to authentic amyloid plaques.

Alzheimer Disease↗
PubMed1995

Rational design, synthesis, and biological evaluation of novel growth hormone releasing factor analogues.

Since its initial discovery in 1982, growth hormone-releasing factor (GRF) has been the subject of intense investigation. This interest was prompted by the potential application of GRF for stimulating growth in dwarf humans and for performance enhancement in livestock. Substantial research has been focused upon the development of potent, long-acting analogs as therapeutics. Herein is described a summary of the cumulative efforts of various laboratories endeavoring in this quest. The rationale utilized in GRF analog development is discussed: 1) determination of bioactive core, 2) evaluation of secondary structure, and 3) elucidation of degradation pathways (chemical and enzymatic). Using this information, several series of linear (unnatural and natural sequence) and cyclic GRF analogs were designed, synthesized, and evaluated. Stimulated by the constraints of commercial production, innovative, alternative methods of synthesis were explored: solid-phase, solution-phase, enzymatic, and recombinant. To date, the most promising candidate for drug development is [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH. This natural sequence analog, consisting of rodent and human sequences, incorporates the bioactive core, preferred secondary structure, resistance to chemical and enzymatic degradation; with the added benefit of amenability to large-scale recombinant synthesis.

Amino Acid Sequence↗
PubMed1994

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences.

Native human GRF(1-44)-NH2(hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3. The hGRF44 is also subject to chemical rearrangement (Asn8-->Asp8, beta-Asp8 via aminosuccinimide formation) and oxidation [Met27-->Met(O)27] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop long-acting GRF analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Ala15 substitution (for Gly15), previously demonstrated to enhance receptor binding affinity, would be predicted to improve GRF analogue potency. Substitution of [His1,Val2]-(from the mouse GRF sequence) for [Tyr1,Ala2]-(human sequence) in [Ala15,Leu27]hGRF(1-32)-OH analogues completely inhibited (24-h incubation) DPP-IV cleavage and greatly increased plasma stability in vitro. Additional substitution of Thr8 (mouse GRF sequence), Ser8 (rat GRF sequence), or Gln8 (not naturally occurring) for Asn8 (human GRF sequence) resulted in analogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resistant hGRF(1-32)-OH analogues, containing His1, Val2, Thr/Gln8, Ala15, and Leu27 replacements, were then bioassayed for growth hormone (GH)-releasing activity in vitro (rat pituitary cell culture) and in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1-32)-OH analogues. In vitro, these analogues were approximately threefold more potent than hGRF44, whereas in vivo they were eleven- to thirteenfold more potent.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), from the precursor, [Ala15,29]-GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala15]-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease. In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3. GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 degrees C). The extent of conversion of 2 into 4 is limited by proteolyses at Asp3-Ala4 and Asp25-Ile26. However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R. A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH2Tyr-D-Ala-Asp(OH)-OR (3) [R = CH3CH2- (3a), CH3- (3b), ClCH2CH2- (3c), C6H5CH2- (3d), 4-NO2C6H4CH2- (3e)] revealed that rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (Vmax/Km) of GSE for the tripeptide ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1994

Pegylated peptides. II. Solid-phase synthesis of amino-, carboxy- and side-chain pegylated peptides.

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.

Amino Acid Sequence↗
PubMed1992

Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor.

A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)]. The main focus of this report was to develop conditions to use the V8 protease-catalyzed coupling while avoiding a competing cleavage of the proteolytically-sensitive Asp25-Ile26 bond in GRF. Conversion of 2 to 4 in couplings employing the alpha-ethyl ester of the acyl component 3a was limited to about 60% by competing proteolysis at Asp25-Ile26. This system was adequate for preparing, isolating, and fully characterizing the target analog 4 and identifying the side products. The 4-nitrobenzyl ester 3b proved to be a superior substrate, resulting in 90% conversion of 2 to 4 with no detectable loss to proteolysis and requiring significantly lesser amounts of catalyst. These results demonstrate that enzymatic semisynthesis of a biologically-active peptide amide which contains unnatural amino acids at the N-terminus can be achieved from a biosynthetic precursor in good yield and purity.

Amino Acid Sequence↗
PubMed1992

A sensitive and specific competition microELISA for the immunoactive polypeptide parathymosin and detection of this peptide in porcine tissues.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.25-30 pmol (3-330 ng) of parathymosin and the assay was specific. The related immunoactive polypeptides prothymosin alpha or thymosin alpha 1 showed no cross-reactivity. In spiking experiments the recovery of the assay was found to be greater than 92% at all concentrations tested. The intra-assay variation was 17%, whereas the inter-assay variation was 26%. Using this assay the highest concentration of parathymosin was found in porcine liver, followed by kidney, lung, thymus and spleen. This assay compares favorably with one microELISA and two RIA methods already published, in that it is more sensitive by at least an order of magnitude, and it is simpler and quicker.

Animals↗
PubMed1992

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.

Amino Acid Sequence↗