Molecular Dynamics Simulations on the Bioactive Molecule of hIAPP22-29 (NFGAILSS) and Rational Drug Design.

This chapter includes information about the structure in equilibrium of the bioactive molecule hIAPP22-29 (NFGAILSS). The experimental structure was derived using X-ray and its 2D NOESY NMR experiments in d 6-DMSO and d-HFIP solvents. This molecule contains eight of the ten amino acids of the 20-29 region of the human islet amyloid polypeptide (hIAPP) often referred as the "amyloidogenic core." Amyloid deposits are well-known to cause as many as 20 pathological neurodegenerative disorders such as Alzheimer, Parkinson, Huntington, and Creutzfeldt-Jakob. The experimental structure was relaxed using molecular dynamics (MD) in simulation boxes consisting in DMSO and HFIP; the latter not provided by the applied software. The calculations were performed in GPUs and supercomputers, and some basic scripting is described for reference. The simulations confirmed the inter- and intramolecular forces that led to an "amyloidogenic core" observed from NOE experiments. The results showed that in DMSO and HFIP environment, Phe is not in spatial proximity with Leu or Ile, and this is consistent with an amyloidogenic core. However, in an amphipathic environment such as the model lipid bilayers, this communication is possible and may influence peptide amyloidogenic properties. The knowledge gained through this study may contribute to the rational drug design of novel peptides or organic molecules acting by modifying preventing amyloidogenic properties of the hIAPP peptide.

Development of Peptide-Based Inhibitors of Amylin Aggregation Employing Aromatic and Electrostatic Repulsion.

Human islet amyloid polypeptide (hIAPP) is a 37-residue hormone that is co-stored and co-secreted with insulin. In type 2 diabetes, the polypeptide misfolds to form amyloid plaques in the pancreas. The self-assembly of hIAPP has been linked to the loss of insulin production and ß-cell death. Recent investigations have revealed that soluble oligomers of hIAPP are the cytotoxic species responsible for ß-cell death and not insoluble amyloid fibrils. Compounds that prevent the self-assembly of hIAPP or drive self-assembly to the state of innocuous insoluble amyloid may be of potential therapeutic value. In this report we summarize key methods employed in our efforts to identify peptide-based modulators of amylin self-assembly that utilize π-electronic effects or electrostatic charge repulsion. These peptide-based modulators may serve as lead compounds for the development of more drug-like molecules and demonstrate that tuning π-electron density and employing charged amyloid disrupting elements are viable approaches toward the design of potential amyloid inhibitors.

Conformational Differentiation of α-Cyanohydroxycinnamic Acid Isomers: A Raman Spectroscopic Study.

Two α-cyanohydroxycinnamic acid positional isomers, α-cyano-4-hydroxycinnamic acid (CHCA4) and α-cyano-3-hydroxycinnamic acid (CHCA3), were characterized using Raman spectroscopy. We analyzed the implications of the collected Raman spectral shifts, and verified them through other spectroscopic techniques, to arrive at plausible three dimensional structures of CHCA3 and CHCA4. The positions of these groups were mapped by systematically analyzing the orientation and type of interactions functional groups make in each CHCA isomer. We determined whether or not the carboxylic moieties are forming dimeric links and ascertained the existence of ring-ring π-stacking interactions. We also assessed the nature of the hydrogen bonding between -CN and -OH groups. The results were then taken together to model plausible three dimensional structures for each compound. The data revealed a structure for CHCA4 that matches the published x-ray crystallographic structure. We then applied the same spectral analysis to CHCA3 to reveal its plausible three dimensional structure. The structural details revealed may account for the functional properties of the two α-cyanohydroxycinnamic acid positional isomers.

Peptide Conjugates of Benzene Carboxylic Acids as Agonists and Antagonists of Amylin Aggregation.

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37 residue peptide hormone that is stored and co-secreted with insulin. hIAPP plays a pivotal role in type 2 diabetes and is the major component of amyloid deposits found in the pancreas of patients afflicted with the disease. The self-assembly of hIAPP and the formation of amyloid is linked to the death of insulin producing ß-cells. Recent findings suggest that soluble hIAPP oligomers are the cytotoxic species responsible for ß-cell loss whereas amyloid fibrils themselves may indeed be innocuous. Potential avenues of therapeutic intervention include the development of compounds that prevent hIAPP self-assembly as well as those that reduce or eliminate lag time and rapidly accelerate the formation of amyloid fibrils. Both of these approaches minimize temporal exposure to soluble cytotoxic hIAPP oligomers. Toward this end our laboratory has pursued an electrostatic repulsion approach to the development of potential inhibitors and modulators of hIAPP self-assembly. Peptide conjugates were constructed in which benzene carboxylic acids of varying charge were employed as electrostatic disrupting elements and appended to the N-terminal of the hIAPP22-29 (NFGAILSS) self-recognition sequence. The self-assembly kinetics of conjugates were characterized by turbidity measurements and the structure of aggregates probed by Raman and CD spectroscopy while the morphology was assessed using transmission electron microscopy. Several benzene carboxylic acid peptide conjugates failed to self-assemble and some were found to inhibit the aggregation of full-length amylin while others served to enhance the rate of amyloid formation and/or increase the yield of amyloid produced. Studies reveal that the geometric display of free carboxylates on the benzene ring of the conjugates plays an important role in the activity of conjugates. In addition, a number of free benzene carboxylic acids were found to modulate amylin self-assembly on their own. The results of these investigations confirm the viability of the electrostatic repulsion approach to the modulation of amyloid formation and may aid the design and development of potential therapeutic agents.

Multivalent Peptoid Conjugates Which Overcome Enzalutamide Resistance in Prostate Cancer Cells.

Development of resistance to antiandrogens for treating advanced prostate cancer is a growing concern and extends to recently developed therapeutics, including enzalutamide. Therefore, new strategies to block androgen receptor (AR) function in prostate cancer are required. Here, we report the characterization of a multivalent conjugate presenting two bioactive ethisterone ligands arrayed as spatially defined pendant groups on a peptoid oligomer. The conjugate, named Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure-activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its antiproliferative effect, suggesting that both ethisterone ligand presentation and scaffold characteristics contribute to MPC6 activity. Mechanistically, MPC6 blocked AR coactivator-peptide interaction and prevented AR intermolecular interactions. Protease sensitivity assays suggested that the MPC6-bound AR induced a receptor conformation distinct from that of dihydrotestosterone- or enzalutamide-bound AR. Pharmacologic studies revealed that MPC6 was metabolically stable and displayed a low plasma clearance rate. Notably, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer. Cancer Res; 76(17); 5124-32. ©2016 AACR.

Aromaticity and amyloid formation: effect of π-electron distribution and aryl substituent geometry on the self-assembly of peptides derived from hIAPP(22-29).

A comprehensive investigation of peptides derived from the 22-29 region of human islet amyloid polypeptide (hIAPP) that contain phenylalanine analogs at position 23 with a variety of electron donating and withdrawing groups, along with heteroaromatic surrogates, has been employed to interrogate how π-electron distribution effects amyloid formation. Kinetic aggregation studies using turbidity measurements indicate that electron rich aromatic ring systems consistently abolish the amyloidogenic propensity of hIAPP(22-29). Electron poor systems modulate the rate of aggregation. Raman and Fourier transform infrared spectroscopy confirm the parallel ß-sheet secondary structure of aggregates derived from peptides containing electron poor phenylalanine analogs and provide direct evidence of ring stacking. Transmission electron microscopy confirms the presence of amyloid fibrils. The effect of aryl substituent geometry on peptide self-assembly reveals that the electronic nature of substituents and not their steric profile is responsible for failure of the electron donating group peptides to aggregate. Non-aggregating hIAPP(22-29) peptides were found to inhibit the self-assembly of full-length hIAPP(1-37). The most potent inhibitory peptides contain phenylalanine with the p-amino and p-formamido functionalities. These novel peptides may serve as leads for the development of future aggregation inhibitors. A potential mechanism for inhibition of amylin self-assembly by electron rich (-29) peptides is proposed.

Evidence of π-stacking interactions in the self-assembly of hIAPP(22-29).

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of human islet amyloid polypeptide (hIAPP)22-29 , peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22-29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in ß-sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the CC ring mode (ca. 1600 cm(-1) ) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22-29 . © Proteins 2013. © 2012 Wiley Periodicals, Inc.

Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor.

The spectral properties of the SH2 and active site-directed sequences of the bivalent Src kinase inhibitor Ac-EELL(F5)Phe-(GABA)3-pYEEIE-amide (1) have been determined. Ac-pYEEIE-amide (2) and AcEELL(F5)Phe-amide (3), as well as the amino acids phosphotyrosine (pTyr) and pentafluorophenylalanine (F5)Phe, have been characterized by electronic absorption, fluorescence, and vibrational spectroscopy. Specific and unique marker bands that originate from the phosphate group of pTyr and the fluorinated aromatic ring of (F5)Phe have been identified, with the latter showing some solvent dependence. Peptide 2 was found to have excitation and emission wavelengths emanating from pTyr at 268 and 295 nm, respectively, whereas peptide 3 displayed excitation and emission peaks attributable to (F5)Phe at 274 and 315 nm, respectively. Fourier transform infrared (FT-IR) analysis of the amino acid pTyr identified distinct marker bands at approximately 930, 1090, and 1330 cm(-1) that could be attributed to the phosphate group. These markers were also observed in the IR spectrum of peptide 2. Likewise, peptide 3 displayed a characteristic C-F stretching mode at 961 cm(-1) due to the presence of (F5)Phe, including two C-F reporting ring modes at 1509 and 1527 cm(-1). Identifying and monitoring spectroscopic changes in these marker bands may afford a means to observe the molecular interactions that occur when peptides 1-3 bind to the Src kinase.

Synthesis of Photoactivatable 1,2-O-Diacyl-sn-glycerol Derivatives of 1-L-Phosphatidyl-D-myo-inositol 4,5-Bisphosphate (PtdInsP(2)) and 3,4,5-Trisphosphate (PtdInsP(3)).

Photoactivatable analogues of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P(2) or PtdInsP(2)) and the corresponding 3,4,5-trisphosphate (PtdIns(3,4,5)P(3) or PtdInsP(3)) were prepared from the two chiral precursors, methyl alpha-D-glucopyranoside and 1,2-isopropylidene-sn-glycerol. Two key synthetic transformations included the Ferrier rearrangement reaction to construct the optically-pure inositol skeleton and the sequential acylation of the primary and secondary hydroxyl groups on the glycerol derivatives. The sn-1-O-(6-aminohexanoyl) PtdInsP(2) and PtdInsP(3) derivatives were further modified to contain benzophenone photophores in unlabeled and high specific activity tritium-labeled forms.