Paraoxonase 1 (PON1) is a calcium-dependent hydrolase associated with serum high-density lipoprotein particles. PON1 hydrolyzes some organophosphates (OPs), including some nerve agents, through nucleophilic attack of hydroxide ion (from water) in the active site. Most OPs are hydrolyzed inefficiently. This project seeks to identify nucleophiles that can enhance PON1-mediated OP degradation. A series of novel nucleophiles, substituted phenoxyalkyl pyridinium oximes, has been synthesized which enhance the degradation of surrogates of sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). Two types of in vitro assays have been conducted, a direct assay using millimolar concentrations of substrate with direct spectrophotometric quantitation of a hydrolysis product (4-nitrophenol) and an indirect assay using submicromolar concentrations of substrate with quantitation by the level of inhibition of an exogenous source of acetylcholinesterase from non-hydrolyzed substrate. Neither NIMP nor NEMP is hydrolyzed effectively by PON1 if one of these novel oximes is absent. However, in the presence of eight novel oximes, PON1-mediated degradation of both surrogates occurs. Computational modeling has created a model of PON1 embedded in phospholipid and has indicated general agreement of the binding enthalpies with the relative efficacy as PON1 enhancers. PON1 enhancement of degradation of OPs could be a unique and unprecedented mechanism of antidotal action.
Alzheimer’s Disease is characterized by the formation of amyloid beta (A[Formula: see text] fibril plaques in the brain. These fibrils can be probed by solid state NMR (ssNMR), which leads to an ensemble of configurations that are compatible with the NMR signals. Typically, only the lowest energy conformer is considered in computer simulations that probe the stability of fibrils and their binding with drug candidates. This restriction could produce data that are not physiologically relevant if the NMR entries differ significantly in stability or binding affinities. In order to study this effect, we have investigated the variance in stability between members of NMR ensembles. Our test cases are a patient-derived A[Formula: see text]-fibril model and two in vitro A[Formula: see text]-fibril models from a previous study we performed on comparative stability. The latter two models allow us also to compare different staggering patterns. We observe significant variations in molecular flexibility, compactness and secondary structure, suggesting that the full NMR ensemble must be considered for a physiologically relevant description of A[Formula: see text] fibrils.
Amyloid deposits of light-chain proteins are associated with the most common form of systemic amyloidosis. We have studied the effects of single point mutations on amyloid formation of these proteins using explicit solvent model molecular dynamics simulations. For this purpose, we compare the stability of the wild-type immunoglobulin light-chain protein REI in its native and amyloid forms with that of four mutants: R61N, G68D, D82I, and A84T. We argue that the experimentally observed differences in the propensity for amyloid formation result from two effects. First, the mutant dimers have a lower stability than the wild-type dimer due to increase exposure of certain hydrophobic residues. The second effect is a shift in equilibrium between monomers with amyloid-like structure and such with native structures. Hence, when developing drugs against light-chain associated systemic amyloidosis, one should look for components that either stabilize the dimer by binding to the dimer interface or reduce for the monomers the probability of the amyloid form.
Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.
Purpose: Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radio-therapies, targeted drug delivery is now gaining focus of cancer researchers. The key for success for any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Four over-expressed receptors which can be targeted on the cell surface of PCa cells are: (GnRH), (EGFR), (PSMA), and (uPAR). Thus, we propose to study the binding affinity of tweleve novel designed targeting peptides (three peptides per receptor) for these over-expressed receptors. Initially, we will study the binding affinity using a computerized molecular modeling program. We will corroborate our modeling data by determining the dissociation constants of the peptide-receptor interaction using sensitive experimental tools such as miscroscale thermophoresis. Methods: The newly designed peptides were generated in pdb (protein database) format and the known pdb files of the receptors were collected from protein data bank. Cluspro 2.0, developed by Boston University, is a protein docking server with a molecular modeling program that was used to calculate the binding energies of these peptides to their corresponding receptors. The data obtained was analyzed using Pymol. Results: The molecular docking studies showed that the GnRH tarteting peptide QH10, uPAR targeting peptide CV13, EGFR targeting peptide RL10, and PSMA targeting peptide TH12 have the lowest mean binding energies for their respective top three binding conformations. The calculatd binding energies for the aftermentioed peptides-receptors combination are: -1285 KCal/M, -1029 KCal/M, -1019 KCal/M and -1037 KCal/M, respectively. There results suggested a very high interaction for these newly designed peptides and their corroponding receptors as the typical calculated binding energy for an antigen-antibody interaction is -700 KCal/M. Conclusion: Based on our molecular docking studies, we have determined the best targeting peptides among all the newly desiged peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated using microscale thermophoresis. This work is supported by grant funding from NIH/RCMI G12MD00758 (T. Turner) and NIH/NCI U54 CA118623 (T. Turner & M. Abdalla). Note: This abstract was not presented at the meeting. Citation Format: Mohamed O. Abdalla, Ahmad Bin Salam, Vincent Hembrick, Manikanthan Bhavaraju, Clayton Yates, Jesse Jaynes, Timothy Turner. Molecular modeling studies of novel receptor targeted peptides in the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2015-3694
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