Publications by authors named "Jamie S Simpson"

34 Publications

Targeted delivery of mycophenolic acid to the mesenteric lymph node using a triglyceride mimetic prodrug approach enhances gut-specific immunomodulation in mice.

J Control Release 2021 Apr 17;332:636-651. Epub 2021 Feb 17.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia. Electronic address:

The mesenteric lymph nodes (MLN) are a key site for the generation of adaptive immune responses to gut-derived antigenic material and immune cells within the MLN contribute to the pathophysiology of a range of conditions including inflammatory and autoimmune diseases, viral infections, graft versus host disease and cancer. Targeting immunomodulating drugs to the MLN may thus be beneficial in a range of conditions. This paper investigates the potential benefit of targeting a model immunosuppressant drug, mycophenolic acid (MPA), to T cells in the MLN, using a triglyceride (TG) mimetic prodrug approach. We confirmed that administration of MPA in the TG prodrug form (MPA-TG), increased lymphatic transport of MPA-related species 83-fold and increased MLN concentrations of MPA >20 fold, when compared to MPA alone, for up to 4 h in mice. At the same time, the plasma exposure of MPA and MPA-TG was similar, limiting the opportunity for systemic side effects. Confocal microscopy and flow cytometry studies with a fluorescent model prodrug (Bodipy-TG) revealed that the prodrug accumulated in the MLN cortex and paracortex at 5 and 10 h following administration and was highly associated with B cells and T cells that are found in these regions of the MLN. Finally, we demonstrated that MPA-TG was significantly more effective than MPA at inhibiting CD4 and CD8 T cell proliferation in the MLN of mice in response to an oral ovalbumin antigen challenge. In contrast, MPA-TG was no more effective than MPA at inhibiting T cell proliferation in peripheral LN when mice were challenged via SC administration of ovalbumin. This paper provides the first evidence of an in vivo pharmacodynamic benefit of targeting the MLN using a TG mimetic prodrug approach. The TG mimetic prodrug technology has the potential to benefit the treatment of a range of conditions where aberrant immune responses are initiated in gut-associated lymphoid tissues.
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http://dx.doi.org/10.1016/j.jconrel.2021.02.008DOI Listing
April 2021

The Impact of Conjugation Position and Linker Chemistry on the Lymphatic Transport of a Series of Glyceride and Phospholipid Mimetic Prodrugs.

J Pharm Sci 2021 01 15;110(1):489-499. Epub 2020 Oct 15.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia. Electronic address:

Drug delivery to the lymphatic system is gaining increasing attention, particularly in fields such as immunotherapy where drug access to lymphocytes is central to activity. We have previously described a prodrug strategy that facilitates the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA) via incorporation into intestinal triglyceride transport pathways. The current study explored a series of structurally related glyceride and phospholipid mimetic prodrugs of MPA in an attempt to enhance lymph targeting and to better elucidate the design criteria for lipid mimetic prodrugs. MPA was conjugated to a glyceride or phospholipid backbone at various positions using different spacers employing ester, ether, carbonate and amide bonds. Patterns of prodrug hydrolysis were evaluated in rat digestive fluid, and lymphatic transport and plasma pharmacokinetics were assessed in lymph duct cannulated rats. Prodrugs with different spacers between MPA and the glyceride backbone resulted in up to 70-fold differences in gastrointestinal stability. MPA conjugation at the 2 position of the glyceride backbone and via an ester bond were most effective in promoting lymphatic transport. Phospholipid prodrug derivatives, or glyceride derivatives with MPA attached at the 1 position or when linked via ether, carbonate or amide bonds were poorly incorporated into lymphatic transport pathways.
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http://dx.doi.org/10.1016/j.xphs.2020.10.021DOI Listing
January 2021

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.

Sci Transl Med 2017 05;9(392)

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NKR) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NKR in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NKR endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NKR antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NKR antagonists. These results reveal a critical role for endosomal signaling of the NKR in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.
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http://dx.doi.org/10.1126/scitranslmed.aal3447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034632PMC
May 2017

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1.

PLoS One 2017 27;12(3):e0173436. Epub 2017 Mar 27.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173436PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367682PMC
August 2017

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

J Biomol NMR 2016 11 24;66(3):195-208. Epub 2016 Oct 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met CH assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-C,N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.
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http://dx.doi.org/10.1007/s10858-016-0067-4DOI Listing
November 2016

Glyceride-Mimetic Prodrugs Incorporating Self-Immolative Spacers Promote Lymphatic Transport, Avoid First-Pass Metabolism, and Enhance Oral Bioavailability.

Angew Chem Int Ed Engl 2016 10 2;55(44):13700-13705. Epub 2016 Aug 2.

Drug Delivery, Disposition and Dynamics, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

First-pass hepatic metabolism can significantly limit oral drug bioavailability. Drug transport from the intestine through the lymphatic system, rather than the portal vein, circumvents first-pass metabolism. However, the majority of drugs do not have the requisite physicochemical properties to facilitate lymphatic access. Herein, we describe a prodrug strategy that promotes selective transport through the intestinal lymph vessels and subsequent release of drug in the systemic circulation, thereby enhancing oral bioavailability. Using testosterone (TST) as a model high first-pass drug, glyceride-mimetic prodrugs incorporating self-immolative (SI) spacers, resulted in remarkable increases (up to 90-fold) in TST plasma exposure when compared to the current commercial product testosterone undecanoate (TU). This approach opens new opportunities for the effective development of drugs where oral delivery is limited by first-pass metabolism and provides a new avenue to enhance drug targeting to intestinal lymphoid tissue.
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http://dx.doi.org/10.1002/anie.201604207DOI Listing
October 2016

Lymphatic Transport and Lymphocyte Targeting of a Triglyceride Mimetic Prodrug Is Enhanced in a Large Animal Model: Studies in Greyhound Dogs.

Mol Pharm 2016 10 21;13(10):3351-3361. Epub 2016 Sep 21.

School of Animal and Veterinary Sciences, Charles Sturt University , Boorooma Street, Wagga Wagga, New South Wales 2650, Australia.

In previous studies, a triglyceride (TG) mimetic prodrug of the model immunomodulator mycophenolic acid (MPA) was shown to significantly enhance lymphatic transport of MPA-related species in the rat. The rat gastrointestinal tract, however, is somewhat different from that in higher order species such as dogs and humans and may underestimate lymphatic transport. Here the effectiveness of the prodrug strategy has been examined in conscious greyhound dogs, the GI physiology of which is more representative of that in humans. The bioavailability and lymphatic transport of free MPA and total MPA related materials were examined following oral administration of the parent drug (MPA) and the prodrug (2-MPA-TG) to both thoracic lymph duct cannulated and intact (noncannulated) greyhound dogs. The enrichment of free MPA in lymph nodes and lymph-derived lymphocytes was also determined to examine the efficiency of drug targeting to potential sites of action within the lymph. Via biochemical integration into a series of site-specific metabolic processes, the prodrug markedly increased (288-fold) lymphatic transport of total MPA related material (present as re-esterified 2-MPA-TG) when compared to the parent MPA and the extent of lymphatic transport was significantly greater in the dog (36.4% of the dose recovered in lymph) when compared to the previous data in the rat (13.4% of the dose). Conversion from 2-MPA-TG derivatives to parent MPA occurred in vivo, resulting in a marked increase in MPA concentrations in lymph nodes (5-6-fold) and lymph lymphocytes (21-fold), when compared to animals administered the parent drug. In conclusion, the data demonstrate that the TG prodrug of MPA facilitates efficient delivery of MPA to the lymphatic system in dogs and suggest that the TG prodrug strategy may more effectively facilitate targeted delivery in large animals than in rats.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00195DOI Listing
October 2016

Structure-Activity Studies of β-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1-RON2 Interaction.

J Mol Biol 2016 10 14;428(20):3986-3998. Epub 2016 Jul 14.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing K values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1-RON2 interaction.
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http://dx.doi.org/10.1016/j.jmb.2016.07.001DOI Listing
October 2016

Constitutive Triglyceride Turnover into the Mesenteric Lymph Is Unable to Support Efficient Lymphatic Transport of a Biomimetic Triglyceride Prodrug.

J Pharm Sci 2016 Feb 29;105(2):786-796. Epub 2016 Jan 29.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

The triglyceride (TG) mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) biochemically integrates into intestinal lipid transport and lipoprotein assembly pathways and thereby promotes the delivery of mycophenolic acid (MPA) into the lymphatic system. As lipoprotein (LP) formation occurs constitutively, even in the fasted state, the current study aimed to determine whether lymphatic transport of 2-MPA-TG was dependent on coadministered exogenous lipid. In vitro incubation of the prodrug with rat digestive fluid and in situ intestinal perfusion experiments revealed that hydrolysis and absorption of the prodrug were relatively unaffected by the quantity of lipid in formulations. In vivo studies in rats, however, showed that the lymphatic transport of TG and 2-MPA-TG was significantly higher following administration with higher quantities of lipid and that oleic acid (C18:1) was more effective in promoting prodrug transport than lipids with higher degrees of unsaturation. The recovery of 2-MPA-TG and TG in lymph correlated strongly (R(2) = 0.99) and more than 97% of the prodrug was associated with chylomicrons. Inhibition of LP assembly by Pluronic L81 simultaneously inhibited the lymphatic transport of 2-MPA-TG and TG. In conclusion, although the TG mimetic prodrug effectively incorporates into TG resynthetic pathways, lipid coadministration is still required to support efficient lymphatic transport.
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http://dx.doi.org/10.1002/jps.24670DOI Listing
February 2016

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

Proc Natl Acad Sci U S A 2015 Jun 18;112(22):6979-84. Epub 2015 May 18.

Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Department of Microbiology, Monash University, Clayton, VIC 3168, Australia; Department of Infectious Diseases, Monash University, Melbourne, 3004, Australia; Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3010, Australia

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.
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http://dx.doi.org/10.1073/pnas.1423900112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460473PMC
June 2015

Design, Synthesis, and Biological Evaluation of Tetra-Substituted Thiophenes as Inhibitors of p38α MAPK.

ChemistryOpen 2015 Feb 11;4(1):56-64. Epub 2014 Nov 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University 381 Royal Parade, Parkville, VIC 3052 (Australia).

p38α mitogen-activated protein kinase (MAPK) plays a role in several cellular processes and consequently has been a therapeutic target in inflammatory diseases, cancer, and cardiovascular disease. A number of known p38α MAPK inhibitors contain vicinal 4-fluorophenyl/4-pyridyl rings connected to either a 5- or 6-membered heterocycle. In this study, a small library of substituted thiophene-based compounds bearing the vicinal 4-fluorophenyl/4-pyridyl rings was designed using computational docking as a visualisation tool. Compounds were synthesised and evaluated in a fluorescence polarisation binding assay. The synthesised analogues had a higher binding affinity to the active phosphorylated form of p38α MAPK than the inactive nonphosphorylated form of the protein. 4-(2-(4-fluorophenyl)thiophen-3-yl)pyridine had a K i value of 0.6 μm to active p38α MAPK highlighting that substitution of the core ring to a thiophene retains affinity to the enzyme and can be utilised in p38α MAPK inhibitors. This compound was further elaborated using a substituted phenyl ring in order to probe the second hydrophobic pocket. Many of these analogues exhibited low micromolar affinity to active p38α MAPK. The suppression of neonatal rat fibroblast collagen synthesis was also observed suggesting that further development of these compounds may lead to potential therapeutics having cardioprotective properties.
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http://dx.doi.org/10.1002/open.201402076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380954PMC
February 2015

Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

J Med Chem 2015 Feb 16;58(3):1205-14. Epub 2015 Jan 16.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria 3052, Australia.

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.
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http://dx.doi.org/10.1021/jm501402xDOI Listing
February 2015

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

Angew Chem Int Ed Engl 2015 Feb 30;54(7):2179-84. Epub 2014 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052 (Australia) http://www.pharm.monash.edu.au.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
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http://dx.doi.org/10.1002/anie.201410341DOI Listing
February 2015

Profiling the role of deacylation-reacylation in the lymphatic transport of a triglyceride-mimetic prodrug.

Pharm Res 2015 May 2;32(5):1830-44. Epub 2014 Dec 2.

Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, 3052, Australia.

Purpose: Recent studies have demonstrated the potential for a triglyceride (TG) mimetic prodrug to promote the delivery of mycophenolic acid (MPA) to the lymphatic system. Here, the metabolic pathways that facilitate the lymphatic transport of the TG prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) were examined to better inform the design of next generation prodrugs.

Methods: In vitro hydrolysis experiments in simulated intestinal conditions and in vivo rat lymphatic transport experiments were conducted in the presence and absence of orlistat and A922500 (inhibitors of lipolysis and TG re-esterification, respectively), to evaluate the importance of 2-MPA-TG digestion and re-esterification of 2-MPA-MG (the 2-monoglyceride derivative) in promoting lymphatic transport.

Results: 2-MPA-TG was rapidly hydrolysed to 2-MPA-MG on incubation with fresh bile and pancreatic fluid (BPF), but not in simulated gastric fluid, heat-inactivated BPF or BPF + orlistat. Orlistat markedly decreased lymphatic transport and systemic exposure of 2-MPA-TG derivatives suggesting that inhibition of pancreatic lipase hindered luminal digestion and absorption of the prodrug. A922500 also significantly decreased lymphatic transport of 2-MPA-TG but redirected MPA to the portal blood, suggesting that hindered re-acylation of 2-MPA-MG resulted in intracellular degradation.

Conclusion: Incorporation into TG deacylation-reacylation pathways is a critical determinant of the utility of lymph directed TG-mimetic prodrugs.
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http://dx.doi.org/10.1007/s11095-014-1579-9DOI Listing
May 2015

Molecular insights into the interaction between Plasmodium falciparum apical membrane antigen 1 and an invasion-inhibitory peptide.

PLoS One 2014 24;9(10):e109674. Epub 2014 Oct 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; Australian Research Council Centre of Excellence for Coherent X-ray Science, Monash University, Parkville, Victoria, Australia.

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1:1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208761PMC
June 2015

Pharmacological characterization and chemical fractionation of a liposterolic extract of saw palmetto (Serenoa repens): effects on rat prostate contractility.

J Ethnopharmacol 2014 Mar 21;152(2):283-91. Epub 2014 Jan 21.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Ethnopharmacological Relevance: Saw palmetto (Serenoa repens) was first used medicinally by native American Indians to treat urological disorders. Nowadays, saw palmetto extracts are widely used in Europe and North America to treat the urinary symptoms associated with benign prostatic hyperplasia even though its mechanisms of action are poorly understood. This study aimed to characterize the bioactive constituents of a lipid extract of saw palmetto that are able to affect contractility of the rat prostate gland. The mechanism of action will also be investigated.

Materials And Methods: A commercially available lipid extract of saw palmetto was subjected to fractionation using normal phase column chromatography. Composition of fractions was assessed by proton nuclear magnetic resonance spectroscopy ((1)H NMR) and mass spectrometry (MS). Contractile activities of these fractions were evaluated pharmacologically using isolated preparations of rat prostate gland and compared to the activity of the crude extract.

Results: Saw palmetto extract inhibited contractions of the rat prostate gland which were consistent with smooth muscle relaxant activity. Only the ethyl acetate fraction resulting from chromatography inhibited contractions of isolated rat prostates similarly to the inhibition produced by the crude lipid extract. Comparison with authentic samples and analysis of NMR data revealed that this bioactivity was due to the fatty acid components present in the ethyl acetate fraction. Bioassay using various pharmacological tools identified multiple contractile mechanisms which were affected by the bioactive constituents.

Conclusion: A fatty acid component of saw palmetto extract causes inhibition of prostatic smooth muscle contractions via a non-specific mechanism.
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http://dx.doi.org/10.1016/j.jep.2013.12.030DOI Listing
March 2014

Targeted delivery of a model immunomodulator to the lymphatic system: comparison of alkyl ester versus triglyceride mimetic lipid prodrug strategies.

J Control Release 2014 Mar 5;177:1-10. Epub 2014 Jan 5.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia. Electronic address:

A lipophilic prodrug approach has been used to promote the delivery of a model immunomodulator, mycophenolic acid (MPA), to the lymphatic system after oral administration. Lymphatic transport was employed to facilitate enhanced drug uptake into lymphocytes, as recent studies demonstrate that targeted drug delivery to lymph resident lymphocytes may enhance immunomodulatory effects. Two classes of lymph-directing prodrugs were synthesised. Alkyl chain derivatives (octyl mycophenolate, MPA-C8E; octadecyl mycophenolate, MPA-C18E; and octadecyl mycophenolamide, MPA-C18AM), to promote passive partitioning into lipids in lymphatic transport pathways, and a triglyceride mimetic prodrug (1,3-dipalmitoyl-2-mycophenoloyl glycerol, 2-MPA-TG) to facilitate metabolic integration into triglyceride deacylation-reacylation pathways. Lymphatic transport, lymphocyte uptake and plasma pharmacokinetics were assessed in mesenteric lymph and carotid artery cannulated rats following intraduodenal infusion of lipid-based formulations containing MPA or MPA prodrugs. Patterns of prodrug hydrolysis in rat digestive fluid, and cellular re-esterification in vivo, were evaluated to examine the mechanisms responsible for lymphatic transport. Poor enzyme stability and low absorption appeared to limit lymphatic transport of the alkyl derivatives, although two of the three alkyl chain prodrugs - MPA-C18AM (6-fold) and MPA-C18E (13-fold) still increased lymphatic drug transport when compared to MPA. In contrast, 2-MPA-TG markedly increased lymphatic drug transport (80-fold) and drug concentrations in lymphocytes (103-fold), and this was achieved via biochemical incorporation into triglyceride deacylation-reacylation pathways. The prodrug was hydrolysed rapidly to 2-mycophenoloyl glycerol (2-MPA-MG) in the presence of rat digestive fluid, and 2-MPA-MG was subsequently re-esterified in the enterocyte with oleic acid (most likely originating from the co-administered formulation) prior to accessing the lymphatics and lymphocytes. Importantly, after administration of 2-MPA-TG, the concentrations of free MPA in the mesenteric lymph nodes were significantly enhanced (up to 28 fold) when compared to animals administered equimolar quantities of MPA, suggesting the efficient conversion of the esterified prodrug back to the pharmacologically active parent drug. The data suggest that triglyceride mimetic prodrugs have potential as a means of enhancing immunotherapy via drug targeting to lymphocytes and lymph nodes.
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http://dx.doi.org/10.1016/j.jconrel.2013.12.031DOI Listing
March 2014

The structure of integrin α1I domain in complex with a collagen-mimetic peptide.

J Biol Chem 2013 Dec 1;288(52):36796-809. Epub 2013 Nov 1.

From Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences and.

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.
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http://dx.doi.org/10.1074/jbc.M113.480251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873540PMC
December 2013

Assignments of human integrin α1I domain in the apo and Mg²⁺ bound states.

Biomol NMR Assign 2014 Apr 22;8(1):117-21. Epub 2013 Jan 22.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC, 3052, Australia.

The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg(2+) bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination.
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http://dx.doi.org/10.1007/s12104-013-9465-7DOI Listing
April 2014

Virtual screening using a conformationally flexible target protein: models for ligand binding to p38α MAPK.

J Comput Aided Mol Des 2012 Apr 20;26(4):409-23. Epub 2012 Apr 20.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC, 3052, Australia.

We have used virtual screening to develop models for the binding of aryl substituted heterocycles to p38α MAPK. Virtual screening was conducted on a number of p38α MAPK crystal structures using a library of 46 known p38α MAPK inhibitors containing a heterocyclic core substituted by pyridine and fluorophenyl rings (structurally related to SB203580) and a set of decoy compounds. Multiple protonation states and tautomers of active and decoy compounds were considered. Each docking model was evaluated using receiver operating characteristic (ROC) curves and enrichment factors. The two best performing single crystal structures were found to be 1BL7 and 2EWA, with enrichment factors of 14.1 and 13.0 at 2% of the virtual screen respectively. Ensembles of up to four receptors of similar conformations were generated, generally giving good or very good performances with high ROC AUCs and good enrichment. The 1BL7-2EWA ensemble was able to outperform each of its constituent receptors and gave high enrichment factors of 17.3, 12.0, 8.0 at 2, 5 and 10% respectively, of the virtual screen. A ROC AUC of 0.94 was obtained for this ensemble. This method may be applied to other proteins where there are a large number of inhibitor classes with different binding site conformations.
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http://dx.doi.org/10.1007/s10822-012-9569-7DOI Listing
April 2012

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

PLoS One 2012 19;7(1):e29444. Epub 2012 Jan 19.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K(d), of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC(50) of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and (15)N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5'-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered--an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including (15)N-(1)H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029444PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261883PMC
June 2012

Synthesis of unsymmetrical 1,1'-disubstituted bis(1,2,3-triazole)s using monosilylbutadiynes.

Org Lett 2011 Feb 12;13(3):537-9. Epub 2011 Jan 12.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

Bis(1,2,3-triazole)s have attracted recent interest as coordinating ligands for transition metals. Here we report a rapid, modular method for the synthesis of 1,1'-disubstituted-4,4'-linked unsymmetrical bis(1,2,3-triazole)s. The method employs sequential copper catalyzed azide-alkyne cycloaddition and deprotection steps on a monosilylbutadiyne. TMS (trimethylsilyl) and TIPS (triisopropylsilyl) were both investigated with TIPS being the preferred protecting group due to increased stability. The reactions were amenable to one-pot synthesis, and an optimized one-pot, three-step procedure was developed.
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http://dx.doi.org/10.1021/ol102852zDOI Listing
February 2011

Ethanol extracts of saw palmetto contain the indirectly acting sympathomimetic: tyramine.

Prostate 2011 Jan;71(1):71-80

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Background: To identify the bioactive components of saw palmetto ethanol extracts that affect contractility in the rat prostate gland.

Methods: A commercially available saw palmetto ethanol extract was lyophilized then subjected to fractionation using silica gel column chromatography. Composition of fractions was assessed by proton nuclear magnetic resonance ((1)H NMR) spectroscopy and mass spectrometry (MS). Contractile activity of these fractions was evaluated pharmacologically using isolated preparations of rat prostate gland and compared to the activity of crude ethanol extract.

Results: Saw palmetto ethanol extract caused contractions of the rat prostate gland which were consistent with indirectly acting sympathomimetic activity. Fractions resulting from chromatography produced contractions of isolated rat prostates that were similar in magnitude to the contractions produced by the crude extracts. Analysis of NMR and mass spectra revealed that this bioactivity was due to tyramine in the active fraction.

Conclusions: Tyramine is present in saw palmetto ethanol extracts and causes indirect α(1)-adrenoceptor mediated contractions via the release of noradrenaline from sympathetic neurons. This has clinical implications, as tyramine interacts with MAO inhibitors to cause hypertensive crisis.
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http://dx.doi.org/10.1002/pros.21223DOI Listing
January 2011

Crystallization and preliminary X-ray analysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 May 30;66(Pt 5):575-8. Epub 2010 Apr 30.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 A, alpha = gamma = 90.0, beta = 100.2 degrees . The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V(M)) of 2.04 A(3) Da(-1) and an estimated solvent content of 39.6%.
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http://dx.doi.org/10.1107/S1744309110010857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864696PMC
May 2010

Structure and function of the oxidoreductase DsbA1 from Neisseria meningitidis.

J Mol Biol 2009 Dec 6;394(5):931-43. Epub 2009 Oct 6.

The Protein Crystallography Unit, Australian Research Council Center of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.

Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-A-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.
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http://dx.doi.org/10.1016/j.jmb.2009.09.065DOI Listing
December 2009

Backbone assignments of the 34 kDa ketopantoate reductase from E. coli.

Biomol NMR Assign 2008 Jun 26;2(1):93-6. Epub 2008 Apr 26.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia.

Ketopantoate reductase is an essential enzyme for pantothenate (vitamin B5) synthesis and a potential antibiotic target. Here we report the 15N and 1HN, 13C', 13C(alpha) and 13C(beta) chemical shift assignments of the 34 kDa ketopantoate reductase in its apo state.
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http://dx.doi.org/10.1007/s12104-008-9093-9DOI Listing
June 2008

The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes.

J Biol Chem 2009 Jun 22;284(26):17835-45. Epub 2009 Apr 22.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
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http://dx.doi.org/10.1074/jbc.M109.011502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719422PMC
June 2009

Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein.

Mol Cell Biochem 2009 Jun 22;326(1-2):87-95. Epub 2009 Jan 22.

Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, 3052, Victoria, Australia.

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.
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http://dx.doi.org/10.1007/s11010-008-0009-xDOI Listing
June 2009

Aromatic chlorination of omega-phenylalkylamines and omega-phenylalkylamides in carbon tetrachloride and alpha,alpha,alpha-trifluorotoluene.

Org Biomol Chem 2006 Jul 8;4(14):2716-23. Epub 2006 Jun 8.

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.

The aromatic halogenation of simple alkylbenzenes with chlorine proceeds smoothly in acetic acid but is much less efficient in less polar solvents. By contrast chlorination of omega-phenylalkylamines, such as 3-phenylpropylamine, occurs readily in either acetic acid, carbon tetrachloride or alpha,alpha,alpha-trifluorotoluene, and in the latter solvents gives high proportions of ortho-chlorinated products. These effects are attributable to the involvement of N-chloroamines as reaction intermediates, with intramolecular delivery of the chlorine electrophile. Omega-phenylalkylamides, such as 3-phenylpropionamide, also easily undergo aromatic chlorination in carbon tetrachloride and alpha,alpha,alpha-trifluorotoluene. These reactions generally show a first-order dependence on the substrate concentration, but not on the amount of chlorine. With carbon tetrachloride, very similar reaction rates are observed with chlorine concentrations ranging from 0.1-1.5 M. In alpha,alpha,alpha-trifluorotoluene, the rates reach a plateau at a chlorine concentration of approximately 0.2 M. These features indicate that the reactions proceed via the formation of intermediates which evidence suggests may be the corresponding O-chloroimidates. Irrespective of the mechanistic details, the reactions are remarkably rapid, being faster than analogous reactions in acetic acid and three to four orders of magnitude more rapid than reactions of simple alkylbenzenes in carbon tetrachloride. Therefore, chlorination of the amines and amides may be accomplished without the need for highly polar solvents, added catalysts or large excesses of chlorine, which are often employed for electrophilic aromatic substitutions. Although the use of carbon tetrachloride is becoming increasingly impractical due to environmental concerns, the trifluorotoluene is a suitable alternative.
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http://dx.doi.org/10.1039/b605010gDOI Listing
July 2006

Inhibition of peptidylglycine alpha-amidating monooxygenase by exploitation of factors affecting the stability and ease of formation of glycyl radicals.

J Am Chem Soc 2004 Oct;126(41):13306-11

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.

Peptidylglycine alpha-amidating monooxygenase catalyzes the biosynthesis of peptide hormones through radical cleavage of the C-terminal glycine residues of the corresponding prohormones. We have correlated ab initio calculations of radical stabilization energies and studies of free radical brominations with the extent of catalysis displayed by peptidylglycine alpha-amidating monooxygenase, to identify classes of inhibitors of the enzyme. In particular we find that, in closely related systems, the substitution of glycolate for glycine reduces the calculated radical stabilization energy by 34.7 kJ mol(-1), decreases the rate of bromination with N-bromosuccinimide at reflux in carbon tetrachloride by a factor of at least 2000, and stops catalysis by the monooxygenase, while maintaining binding to the enzyme.
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http://dx.doi.org/10.1021/ja046204nDOI Listing
October 2004