Publications by authors named "Sheena McGowan"

73 Publications

High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A Streptococcus.

Mol Microbiol 2021 Mar 23. Epub 2021 Mar 23.

Biomedicine Discovery Institute, Department of Microbiology, Monash University, Melbourne, VIC, Australia.

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.
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http://dx.doi.org/10.1111/mmi.14719DOI Listing
March 2021

Active site metals mediate an oligomeric equilibrium in M17 aminopeptidases.

J Biol Chem 2020 Dec 10. Epub 2020 Dec 10.

Monash University, Australia.

M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel anti-malarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species, that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryo-electron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer  dimer  tetramer  hexamer, which becomes directional towards the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.
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http://dx.doi.org/10.1074/jbc.RA120.016313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948507PMC
December 2020

A Structure-Activity Relationship Study of Novel Hydroxamic Acid Inhibitors around the S1 Subsite of Human Aminopeptidase N.

ChemMedChem 2021 Jan 22;16(1):234-249. Epub 2020 Oct 22.

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

Aminopeptidase N (APN/CD13) is a zinc-dependent ubiquitous transmembrane ectoenzyme that is widely present in different types of cells. APN is one of the most extensively studied metalloaminopeptidases as an anti-cancer target due to its significant role in the regulation of metastasis and angiogenesis. Previously, we identified a potent and selective APN inhibitor, N-(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (3). Herein, we report the further modifications performed to explore SAR around the S1 subsite of APN and to improve the physicochemical properties. A series of hydroxamic acid analogues were synthesised, and the pharmacological activities were evaluated in vitro. N-(1-(3'-Fluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-(methylsulfonamido)benzamide (6 f) was found to display an extremely potent inhibitory activity in the sub-nanomolar range.
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http://dx.doi.org/10.1002/cmdc.202000527DOI Listing
January 2021

X-ray crystal structure and specificity of the Toxoplasma gondii ME49 TgAPN2.

Biochem J 2020 10;477(19):3819-3832

Biomedicine Discovery Institute, Department of Microbiology, Monash University Clayton, Melbourne, VIC 3800, Australia.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii that currently has few therapeutic options. The M1 aminopeptidase enzymes have been shown to be attractive targets for anti-parasitic agents and/or vaccine candidates, suggesting potential to re-purpose inhibitors between parasite M1 aminopeptidase targets. The M1 aminopeptidase TgAPN2 has been suggested to be a potential new drug target for toxoplasmosis. Here we investigate the structure and function of TgAPN2, a homologue of the antimalarial drug target PfA-M1, and evaluate the capacity to use inhibitors that target PfA-M1 against TgAPN2. The results show that despite a similar overall fold, the TgAPN2 has a unique substrate specificity and inhibition profile. Sequence and structure differences are investigated and show how comparative structure-activity relationships may provide a route to obtaining potent inhibitors of TgAPN2.
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http://dx.doi.org/10.1042/BCJ20200569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557147PMC
October 2020

Driving antimalarial design through understanding of target mechanism.

Biochem Soc Trans 2020 10;48(5):2067-2078

Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.

Malaria continues to be a global health threat, affecting approximately 219 million people in 2018 alone. The recurrent development of resistance to existing antimalarials means that the design of new drug candidates must be carefully considered. Understanding of drug target mechanism can dramatically accelerate early-stage target-based development of novel antimalarials and allows for structural modifications even during late-stage preclinical development. Here, we have provided an overview of three promising antimalarial molecular targets, PfDHFR, PfDHODH and PfA-M1, and their associated inhibitors which demonstrate how mechanism can inform drug design and be effectively utilised to generate compounds with potent inhibitory activity.
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http://dx.doi.org/10.1042/BST20200224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609028PMC
October 2020

Reply to: Caution is warranted in using cephamycin antibiotics against recurrent Clostridioides difficile infection.

Nat Microbiol 2020 02 27;5(2):237-238. Epub 2020 Jan 27.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia.

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http://dx.doi.org/10.1038/s41564-019-0662-8DOI Listing
February 2020

Strategies for Increasing Protein Stability.

Methods Mol Biol 2020 ;2073:163-181

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

The stability of wild-type proteins is often a hurdle to their practical use in research, industry, and medicine. The route to engineering stability of a protein of interest lies largely with the available data. Where high-resolution structural data is available, rational design, based on fundamental principles of protein chemistry, can improve protein stability. Recent advances in computational biology and the use of nonnatural amino acids have also provided novel rational methods for improving protein stability. Likewise, the explosion of sequence and structural data available in public databases, in combination with improvements in freely available computational tools, has produced accessible phylogenetic approaches. Trawling modern sequence databases can identify the thermostable homologs of a target protein, and evolutionary data can be quickly generated using available phylogenetic tools. Grafting features from those thermostable homologs or ancestors provides stability improvement through a semi-rational approach. Further, molecular techniques such as directed evolution have shown great promise in delivering designer proteins. These strategies are well documented and newly accessible to the molecular biologist, allowing for rapid enhancements of protein stability.
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http://dx.doi.org/10.1007/978-1-4939-9869-2_10DOI Listing
January 2021

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Nat Microbiol 2019 12 12;4(12):2237-2245. Epub 2019 Aug 12.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.
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http://dx.doi.org/10.1038/s41564-019-0519-1DOI Listing
December 2019

Crystal structure of the inhibitor-free form of the serine protease kallikrein-4.

Acta Crystallogr F Struct Biol Commun 2019 Aug 16;75(Pt 8):543-546. Epub 2019 Jul 16.

Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, 23 Innovation Walk, Clayton, VIC 3800, Australia.

Kallikrein 4 (KLK4) is a serine protease that is predominantly expressed in the prostate and is overexpressed in prostate cancer. As such, it has gained attention as an attractive target for prostate cancer therapeutics. Currently, only liganded structures of KLK4 exist in the Protein Data Bank. Until now, inferences about the subtle structural changes in KLK4 upon ligand binding have been made by comparison to other liganded forms, rather than to an apo form. In this study, an inhibitor-free form of KLK4 was crystallized. The crystals obtained belonged to space group P1, contained four molecules in the asymmetric unit and diffracted to 1.64 Å resolution. Interestingly, a nonstandard rotamer of the specificity-determining residue Asp189 was observed in all chains. This model will provide a useful unliganded structure for the future structure-guided design of KLK4 inhibitors.
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http://dx.doi.org/10.1107/S2053230X19009610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688662PMC
August 2019

Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions.

J Med Chem 2019 08 18;62(15):7185-7209. Epub 2019 Jul 18.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology , Monash University , Clayton Campus , Clayton , VIC 3800 , Australia.

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite M1 aminopeptidase (A-M1). Herein, we describe the rationale that underpins the repurposing of A-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. -(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide () proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound may be a useful lead for the development of anticancer agents.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00757DOI Listing
August 2019

Reactive centre loop dynamics and serpin specificity.

Sci Rep 2019 03 7;9(1):3870. Epub 2019 Mar 7.

Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3800, Australia.

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.
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http://dx.doi.org/10.1038/s41598-019-40432-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405850PMC
March 2019

M17 aminopeptidases diversify function by moderating their macromolecular assemblies and active site environment.

Biochimie 2019 Nov 14;166:38-51. Epub 2019 Jan 14.

Biomedicine Discovery Institute, Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia. Electronic address:

The family of M17 aminopeptidases (alias 'leucine aminopeptidases', M17-LAPs) utilize a highly conserved hexameric structure and a binuclear metal center to selectively remove N-terminal amino acids from short peptides. However, M17-LAPs are responsible for a wide variety of functions that are seemingly unrelated to proteolysis. Herein, we aimed to investigate the myriad of functions attributed to M17. Further, we attempted to differentiate between the different molecular mechanisms that allow the conserved hexameric structure of an M17-LAP to mediate such diverse functions. We have provided an overview of research that identifies precise physiological roles of M17-LAPs, and the distinct mechanisms by which the enzymes moderate those roles. The review shows that the conserved hexameric structure of the M17-LAPs has an extraordinary capability to moderate different molecular mechanisms. We have broadly categorized these mechanisms as 'aminopeptidase-based', which include the characteristic proteolysis reactions, and 'association-driven', which involves moderation of the molecule's macromolecular assembly and higher order complexation events. The different molecular mechanisms are capable of eliciting very different cellular outcomes, and must be regarded as distinct when the physiological roles of this large and important family are considered.
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http://dx.doi.org/10.1016/j.biochi.2019.01.007DOI Listing
November 2019

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

ChemMedChem 2019 03 13;14(5):603-612. Epub 2019 Feb 13.

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

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.
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http://dx.doi.org/10.1002/cmdc.201800802DOI Listing
March 2019

Hydroxamic Acid Inhibitors Provide Cross-Species Inhibition of Plasmodium M1 and M17 Aminopeptidases.

J Med Chem 2019 01 4;62(2):622-640. Epub 2019 Jan 4.

Department of Microbiology, Biomedicine Discovery Institute , Monash University, Clayton , Melbourne , VIC 3800 , Australia.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum ( Pf-M1 and Pf-M17) and P. vivax ( Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1' pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01310DOI Listing
January 2019

Mapping the Pathway and Dynamics of Bestatin Inhibition of the Plasmodium falciparum M1 Aminopeptidase PfA-M1.

ChemMedChem 2018 12 9;13(23):2504-2513. Epub 2018 Nov 9.

Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

The M1 metallo-aminopeptidase from Plasmodium falciparum, PfA-M1, is an attractive drug target for the design of new antimalarials. Bestatin, a broad-spectrum metalloprotease inhibitor, is a moderate inhibitor of PfA-M1, and has been used to provide structure-activity relationships to inform drug design. The crystal structure of PfA-M1 with bestatin bound within its active site has been determined; however, dynamics of the inhibitor and the association or dissociation pathway have yet to be characterized. Here we present an all-atom molecular dynamics study where we have generated a hidden Markov state model from 2.3 μs of molecular dynamics simulation. Our hidden Markov state model identifies five macrostates that clearly show the events involved in bestatin dissociation from the PfA-M1 active site. The results show for the first time that bestatin can escape the substrate specificity pockets of the enzyme, primarily due to weak interactions within the pockets. Our approach identifies relevant conformational sampling of the inhibitor inside the enzyme and the protein dynamics that could be exploited to produce potent and selective inhibitors that can differentiate between similar members of the M1 aminopeptidase superfamily.
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http://dx.doi.org/10.1002/cmdc.201800563DOI Listing
December 2018

Catalytic diversity and cell wall binding repeats in the phage-encoded endolysins.

Mol Microbiol 2018 12 13;110(6):879-896. Epub 2018 Nov 13.

Biomedicine Discovery Institute, Department of Microbiology, Monash University, Victoria, 3800, Australia.

Bacteriophage-encoded endolysins can recognize and bind specific bacteria, and act to cleave the glycosidic and/or amide bonds in the peptidoglycan (PG) bacterial cell wall. Cleavage of the cell wall generally results in the death of the bacteria. Their utility as bacteriolytic agents could be exploited for human and veterinary medicines as well as various biotechnological applications. As interest grows in the commercial uses of these proteins, there has been much effort to successfully employ rational design and engineering to produce endolysins with bespoke properties. In this review, we interrogate the current structural data and identify structural features that would be of benefit to engineering the activity and specificity of phage endolysins. We show that the growing body of structural data can be used to predict catalytic residues and mechanism of action from sequences of hypothetical endolysins, and probe the importance of secondary structure repeats in bacterial cell wall-binding domains.
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http://dx.doi.org/10.1111/mmi.14134DOI Listing
December 2018

Clostridium sordellii outer spore proteins maintain spore structural integrity and promote bacterial clearance from the gastrointestinal tract.

PLoS Pathog 2018 04 18;14(4):e1007004. Epub 2018 Apr 18.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia.

Bacterial spores play an important role in disease initiation, transmission and persistence. In some species, the exosporium forms the outermost structure of the spore and provides the first point of contact between the spore and the environment. The exosporium may also be involved in spore adherence, protection and germination. Clostridium sordellii is a highly lethal, spore forming pathogen that causes soft-tissue infections, enteritis and toxic-shock syndrome. Despite the importance of C. sordellii spores in disease, spore proteins from this bacterium have not been defined or interrogated functionally. In this study, we identified the C. sordellii outer spore proteome and two of the identified proteins, CsA and CsB, were characterised using a genetic and phenotypic approach. Both proteins were essential for the correct formation and positioning of the C. sordellii spore coat and exosporium. The absence of CsA reduced sporulation levels and increased spore sensitivity to heat, sodium hydroxide and hydrochloric acid. By comparison, CsB was required for normal levels of spore adherence to cervical, but not vaginal, cells, with csB mutant spores having increased adherence properties. The establishment of a mouse infection model of the gastrointestinal tract for C. sordellii allowed the role of CsA and CsB to be interrogated in an infected host. Following the oral administration of spores to mice, the wild-type strain efficiently colonized the gastrointestinal tract, with the peak of bacterial numbers occurring at one day post-infection. Colonization was reduced by two logs at four days post-infection. By comparison, mice infected with the csB mutant did not show a reduction in bacterial numbers. We conclude that C. sordellii outer spore proteins are important for the structural and functional integrity of spores. Furthermore, outer spore proteins are required for wild-type levels of colonization during infection, possibly as a result of the role that the proteins play in spore structure and morphology.
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http://dx.doi.org/10.1371/journal.ppat.1007004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927469PMC
April 2018

Long-term outcome of thyrotoxicosis in childhood and adolescence in the west of Scotland: the case for long-term antithyroid treatment and the importance of initial counselling.

Arch Dis Child 2018 07 21;103(7):637-642. Epub 2017 Dec 21.

Child Health Section, Glasgow University School of Medicine, Royal Hospital for Sick Children, Glasgow, UK.

Background: Thyrotoxicosis is both rarer and more severe in children than in adults, rendering management difficult and often unsatisfactory.

Objective: To ascertain outcome in a geographically defined area of Scotland between 1989 and 2014.

Method: Retrospective case note review with follow-up questionnaire to family doctors for patients with Graves' disease and Hashimoto's thyroiditis.

Results: Sixty-six patients (58 females:8 males) comprising 53 with Graves' disease and 13 with Hashimoto's thyroiditis were diagnosed at median 10.4 (2.9-15.8) years and followed up for 11.8 (2.6-30.2) years. Antithyroid drug (ATD) therapy was stopped electively in 35 patients after 4.5 (1.5-8.6) years, resulting in remission in 10/13 Hashimoto's thyroiditis and 10/22 Graves' disease. Side effects occurred in 12 patients receiving carbimazole, six of whom changed to propylthiouracil; no adverse events occurred in the latter patients.Second-line therapy was given to 37 patients (34 with Graves' disease), comprising radioiodine (22) at 15.6 (9.3-24.4) years for relapse (6), poor control/adherence (14) or electively (2); and surgery (16) at 12 (6.4-21.3) years for relapse (4), poor control/adherence (5) and electively (7). Adherence problems with thyroxine replacement were reported in 10/33 patients in adulthood.

Conclusions: Hashimoto's thyroiditis should be distinguished from Graves' disease at diagnosis since the prognosis for remission is better. Remission rates for Graves' disease are low (10/53 patients), time to remission variable and adherence with both ATD and thyroxine replacement often problematic. We recommend (a) the giving of long-term ATD rather than a fixed course of treatment in GD and (b) meticulous and realistic counselling of families from the time of diagnosis onwards.
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http://dx.doi.org/10.1136/archdischild-2017-313454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047164PMC
July 2018

Generation of AMBER force field parameters for zinc centres of M1 and M17 family aminopeptidases.

J Biomol Struct Dyn 2018 Aug 28;36(10):2595-2604. Epub 2017 Aug 28.

a Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute , Monash University , Clayton , Victoria 3800 , Australia.

The M1 and M17 aminopeptidases are metallo-exopeptidases that rely on the presence of divalent cations, usually zinc, in their active site for proteolytic activity. They are from separate protease superfamilies, however, members often have overlapping substrate specificity. Inhibitors of one or both enzymes can be used to modulate hypertension, reduce proliferation of certain types of cancers and control malaria parasites. Current inhibitors act to chelate the zinc ions in the active site, locking the enzymes in an inactive transition state. We were interested in using a computational approach to understand the structure and dynamics of the M1 and M17 aminopeptidases, however, the presence of the essential metal ions in the proteases presents a challenge to classical molecular dynamics (MD) simulation. The zinc amber force field does not contain applicable descriptions of the zinc coordination environment present in either of these two protease families. To provide tools for the study of these two enzymes, we have used the metal centre parameter builder to generate new hybrid bonded/nonbonded force field (FF) parameters to correctly describe the active site architecture for each enzyme. The new parameters were evaluated by fitting the normal mode frequencies of molecular mechanics to the quantum mechanics frequencies and validated by performing short MD simulations. The new FF parameters now enable more accurate and reliable MD simulations for any member of the M1 or M17 aminopeptidase superfamilies.
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http://dx.doi.org/10.1080/07391102.2017.1364669DOI Listing
August 2018

Crystal structure of a β-aminopeptidase from an Australian Burkholderia sp.

Acta Crystallogr F Struct Biol Commun 2017 07 17;73(Pt 7):386-392. Epub 2017 Jun 17.

Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Melbourne, VIC 3800, Australia.

β-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β-amino acids from synthetic β-peptides. β-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Å and showed a tetrameric assembly typical of the β-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a β-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known β-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.
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http://dx.doi.org/10.1107/S2053230X17007737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505242PMC
July 2017

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.

J Mol Biol 2017 03 8;429(6):836-846. Epub 2017 Feb 8.

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia. Electronic address:

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.
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http://dx.doi.org/10.1016/j.jmb.2017.02.003DOI Listing
March 2017

M1 aminopeptidases as drug targets: broad applications or therapeutic niche?

FEBS J 2017 05 3;284(10):1473-1488. Epub 2017 Feb 3.

Biomedicine Discovery Institute, Department of Microbiology, Monash University, Melbourne, Vic., Australia.

M1 aminopeptidase enzymes are a diverse family of metalloenzymes characterized by conserved structure and reaction specificity. Excluding viruses, M1 aminopeptidases are distributed throughout all phyla, and have been implicated in a wide range of functions including cell maintenance, growth and development, and defense. The structure and catalytic mechanism of M1 aminopeptidases are well understood, and make them ideal candidates for the design of small-molecule inhibitors. As a result, many research groups have assessed their utility as therapeutic targets for both infectious and chronic diseases of humans, and many inhibitors with a range of target specificities and potential therapeutic applications have been developed. Herein, we have aimed to address these studies, to determine whether the family of M1 aminopeptidases does in fact present a universal target for the treatment of a diverse range of human diseases. Our analysis indicates that early validation of M1 aminopeptidases as therapeutic targets is often overlooked, which prevents the enzymes from being confirmed as drug targets. This validation cannot be neglected, and needs to include a thorough characterization of enzymes' specific roles within complex physiological pathways. Furthermore, any chemical probes used in target validation must be carefully designed to ensure that specificity over the closely related enzymes has been achieved. While many drug discovery programs that target M1 aminopeptidases remain in their infancy, certain inhibitors have shown promise for the treatment of a range of conditions including malaria, hypertension, and cancer.
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http://dx.doi.org/10.1111/febs.14009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164018PMC
May 2017

X-ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2.

Proteins 2017 05 3;85(5):945-950. Epub 2017 Mar 3.

Infection and Immunity Program, Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria, 3800.

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25227DOI Listing
May 2017

Smoothing a rugged protein folding landscape by sequence-based redesign.

Sci Rep 2016 Sep 26;6:33958. Epub 2016 Sep 26.

Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036219PMC
http://dx.doi.org/10.1038/srep33958DOI Listing
September 2016

Circumventing the stability-function trade-off in an engineered FN3 domain.

Protein Eng Des Sel 2016 Nov;29(11):541-550

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.

The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.
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http://dx.doi.org/10.1093/protein/gzw046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081044PMC
November 2016

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

PLoS Pathog 2016 07 28;12(7):e1005763. Epub 2016 Jul 28.

Department of Biochemistry and Molecular Biology and Huck Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania, United States of America.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.
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http://dx.doi.org/10.1371/journal.ppat.1005763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965013PMC
July 2016

Structure and substrate fingerprint of aminopeptidase P from Plasmodium falciparum.

Biochem J 2016 10 26;473(19):3189-204. Epub 2016 Jul 26.

Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia.

Malaria is one of the world's most prevalent parasitic diseases, with over 200 million cases annually. Alarmingly, the spread of drug-resistant parasites threatens the effectiveness of current antimalarials and has made the development of novel therapeutic strategies a global health priority. Malaria parasites have a complicated lifecycle, involving an asymptomatic 'liver stage' and a symptomatic 'blood stage'. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP). We developed a novel platform to examine the substrate fingerprint of APP from Plasmodium falciparum (PfAPP) and to show that it can catalyze the removal of any residue immediately prior to a proline. Further, we have determined the crystal structure of PfAPP and present the first examination of the 3D structure of this essential malarial enzyme. Together, these analyses provide insights into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics.
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http://dx.doi.org/10.1042/BCJ20160550DOI 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

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions.

Eur J Med Chem 2016 Mar 13;110:43-64. Epub 2016 Jan 13.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville, VIC 3052, Australia. Electronic address:

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. Plasmodium falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics.
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http://dx.doi.org/10.1016/j.ejmech.2016.01.015DOI Listing
March 2016

Stonefish toxin defines an ancient branch of the perforin-like superfamily.

Proc Natl Acad Sci U S A 2015 Dec 1;112(50):15360-5. Epub 2015 Dec 1.

Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia;

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.
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http://dx.doi.org/10.1073/pnas.1507622112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687532PMC
December 2015