Tatiana P. Soares da Costa

Antibacterial silver and gold complexes of imidazole and 1,2,4-triazole derived N-heterocyclic carbenes.

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2DT01657E

Abstract: A series of gold(I) (4a-4h, 5a-5b) and silver(I) (3a-3h) complexes of 1,2,4-triazolylidene and imidazolylidene based N-heterocyclic carbene ligands were prepared and the antibacterial activities of these complexes have been evaluated. The complexes were characterised using

Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2

Publisher: Frontiers Media SA

Date: 25-10-2021

DOI: 10.3389/FIMMU.2021.666813

Abstract: FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab’) 2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

Pursuing DHDPS: an enzyme of unrealised potential as a novel antibacterial target

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C9MD00107G

Abstract: DHDPS represents a novel enzyme target for the development of new antibiotics to combat multidrug resistance.

Synthesis, conformational analysis and antibacterial activity of Au(i)–Ag(i) and Au(i)–Hg(ii) heterobimetallic N-heterocyclic carbene complexes

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0DT02225J

Abstract: A family heterobimetallic Au( i )–Ag( i ) and Au( i )–Hg( ii ) complexes of bis-N-heterocyclic carbene ligands been prepared and their antibacterial properties evaluated.

New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.BIOCHI.2009.12.004

Abstract: Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (DeltaH = -44.5 +/- 0.1 kJ mol(-1)) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (DeltaH = -2.8 +/- 0.1 kJ mol(-1)) formed through the reduction of the intermediate pyruvyl-Schiff-base complex. The binding interaction of (S)-lysine was characterised as a cooperative event in which an entropic pre-organisation step (TDeltaS = 17.6 +/- 1.1 kJ mol(-1)) precedes a secondary enthalpic association (DeltaH = -21.6 +/- 0.2 kJ mol(-1)). This allosteric association was determined to be of a mixed competitive nature in which heterotropic ligand cooperativity was observed to subtly influence the binding events. These results offer new insights into the inhibition of this enzyme, a validated antibiotic target.

Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes

Publisher: Springer Science and Business Media LLC

Date: 03-01-2017

DOI: 10.1038/SREP39277

Abstract: Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes , as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes , providing an excellent platform for the rational design of novel inhibitors. By comparing the structure of L. monocytogenes FabF with other published bacterial FabF structures in complex with known inhibitors and substrates, we highlight conformational changes within the active site, which will need to be accounted for during drug design and virtual screening studies. This high-resolution structure of FabF represents an important step in the development of new classes of antimicrobial agents targeting FabF for the treatment of listeriosis.

Heterocyclic acyl-phosphate bioisostere-based inhibitors of Staphylococcus aureus biotin protein ligase

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.BMCL.2014.08.030

Abstract: Inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL) are generated by replacing the acyl phosphate group of biotinyl-5'-AMP with either a 1,2,3-triazole (see 5/10a/10b) or a 1,2,4-oxadiazole (see 7) bioisostere. Importantly, the inhibitors are inactive against the human BPL. The nature of the 5-substituent in the component benzoxazolone of the optimum 1,2,3-triazole series is critical to activity, where this group binds in the ATP binding pocket of the enzyme.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency

Publisher: Springer Science and Business Media LLC

Date: 06-09-2012

DOI: 10.1007/S00109-011-0811-X

Abstract: Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Mis‐annotations of a promising antibiotic target in high‐priority gram‐negative pathogens

Publisher: Wiley

Date: 24-01-2020

DOI: 10.1002/1873-3468.13733

Abstract: The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7-residue sequence motif to identify mis-annotation of multiple DHDPS genes in the high-priority Gram-negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis-annotations using a combination of enzyme kinetics and X-ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens. PDB ID: 6UE0.

Towards novel herbicide modes of action by inhibiting lysine biosynthesis in plants

Publisher: eLife Sciences Publications, Ltd

Date: 27-07-2021

DOI: 10.7554/ELIFE.69444

Abstract: Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides with novel modes of action are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. The first class of plant DHDPS inhibitors with micromolar potency against Arabidopsis thaliana DHDPS was identified using a high-throughput chemical screen. We determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. The inhibitors also attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide-resistant weeds.

Biotin analogues with antibacterial activity are potent inhibitors of biotin protein ligase

Publisher: American Chemical Society (ACS)

Date: 24-05-2012

DOI: 10.1021/ML300106P

A dual-target herbicidal inhibitor of lysine biosynthesis

Publisher: eLife Sciences Publications, Ltd

Date: 20-06-2022

DOI: 10.7554/ELIFE.78235

Abstract: Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.

Homodimerization attenuates the anti-inflammatory activity of interleukin-37

Publisher: American Association for the Advancement of Science (AAAS)

Date: 24-02-2017

DOI: 10.1126/SCIIMMUNOL.AAJ1548

Abstract: Interleukin-37 homodimerization regulates its anti-inflammatory activity.

Acceptance of clickers in a large multimodal biochemistry class as determined by student evaluations of teaching: Are they just an annoying distraction for distance students?

Publisher: Wiley

Date: 30-10-2016

DOI: 10.1002/BMB.20917

Abstract: A student response system (clickers) was introduced into a second year introductory biochemistry class to improve student engagement and performance. The class was delivered in both internal and distance education (DE) modes, with the DE students receiving recordings of the lectures (including clicker activities). However, there was concern over the use of clickers in internal classes as it may be alienating or distracting to DE students while reviewing the recordings of these lectures. In order to examine students' attitudes toward clickers, closed- and open-ended questions were examined in the student evaluations of teaching (SET). Understanding attitudes of internal and DE students is especially important as differences may exist between these groups due to the different learning environments they experience. Approximately 45% of students completed the surveys, of which 88%-91% provided written comments. Of the written comments, 18% of DE students and 22% of internal students provided unsolicited comments about clickers. Interestingly, no difference was observed in the themes identified in the comments between cohorts. The key themes included 1) clickers were beneficial for learning (and increased knowledge), 2) clickers were engaging/fun, and 3) clickers could have been used more widely. Overall, based on this study, it was believed that clicker usage was not seen as negative activity by DE students and it was worth continuing to use clickers in teaching the large multimodal class studied here. However, there is a need to investigate the potential of new and emerging technologies to provide more interactive experiences for DE students.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

Publisher: MDPI AG

Date: 24-08-2020

DOI: 10.3390/JOF6030145

Abstract: Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46–50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

A dual-target herbicidal inhibitor of lysine biosynthesis

Publisher: Cold Spring Harbor Laboratory

Date: 04-03-2022

DOI: 10.1101/2022.03.04.482975

Abstract: Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target (Soares da Costa et al., 2021). In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides (Soares da Costa et al., 2021). Here we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.

Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis.

Publisher: Springer Science and Business Media LLC

Date: 05-12-2017

DOI: 10.1007/S12551-017-0350-Y

Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus

Publisher: Elsevier BV

Date: 05-2012

DOI: 10.1074/JBC.M112.356576

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1074/JBC.M115.696591

The First Inhibitor of Meso-Diaminopimelate Biosynthesis with Antibacterial Activity Against Multi-Drug Resistant Bacteria

Publisher: Cold Spring Harbor Laboratory

Date: 11-02-2022

DOI: 10.1101/2022.02.10.480023

Abstract: Antibiotic resistance represents one of the biggest threats to global health. While several of our current antibiotics target the peptidoglycan within the bacterial cell wall, only a fraction of its components has been explored for antibiotic development. A component that remains under-exploited is meso -diaminopimelate ( meso -DAP), a constituent of the cross-linking peptide in Gram-negative bacteria. In this study, we employed a high throughput chemical screen to identify the first inhibitor of meso -DAP biosynthesis with antibacterial activity. Indeed, the compound was shown to have minimum inhibitory concentration values of 8–16 μg/mL against a panel of multi-drug resistant Acinetobacter baumannii strains, including those resistant to the last resort antibiotic carbapenem. Importantly, the compound targets the meso -DAP biosynthesis pathway specifically, with no off-target effects observed in human cell lines, and no resistance exhibited upon continuous treatment, under the conditions tested. Furthermore, we revealed for the first time that meso -DAP biosynthesis inhibition prevents biofilm formation and disrupts established biofilms in A. baumannii . Using a Galleria mellonella model, we showed that this compound improves survival rates against A. baumannii infection by up to 40% relative to the no treatment controls. Lastly, we determined that the inhibitor potentiates the activity of several antibiotic classes, including carbapenems. Thus, this study provides proof-of-concept that meso -DAP biosynthesis represents a promising target for the development of standalone antibacterial agents with a new mode of action as well as adjuvants to be used in combinatorial regimens to rejuvenate our current antibiotic arsenal to combat resistance. Resistance levels to available antibiotics continues to rise, with a growing number of Gram-negative bacterial infections, in particular A. baumannii infections, becoming life-threatening. Despite this, there have been no new classes of antibiotics against Gram-negative bacteria introduced to the market over the last 40 years. Hence, new targets and therapeutics are urgently required to combat these clinically important pathogens. One such target is meso -DAP, a critical component of the cross-linking peptides in the cell walls of Gram-negative bacteria. Here, we describe the first inhibitor of bacterial meso -DAP biosynthesis, with antibacterial activity against multi-drug resistant Gram-negative bacterial strains, including carbapenem-resistant A. baumannii . We also reveal that meso -DAP biosynthesis inhibition affects biofilm stability and potentiates the activity of several antibiotic classes. This study highlights the need to further explore meso -DAP biosynthesis and other unexploited targets in the search for antibiotics with new modes of action.

Structural basis for importin alpha 3 specificity of W proteins in Hendra and Nipah viruses

Publisher: Springer Science and Business Media LLC

Date: 12-09-2018

DOI: 10.1038/S41467-018-05928-5

Abstract: Seven human isoforms of importin α mediate nuclear import of cargo in a tissue- and isoform-specific manner. How nuclear import adaptors differentially interact with cargo harbouring the same nuclear localisation signal (NLS) remains poorly understood, as the NLS recognition region is highly conserved. Here, we provide a structural basis for the nuclear import specificity of W proteins in Hendra and Nipah viruses. We determine the structural interfaces of these cargo bound to importin α1 and α3, identifying a 2.4-fold more extensive interface and 50-fold higher binding affinity for importin α3. Through the design of importin α1 and α3 chimeric and mutant proteins, together with structures of cargo-free importin α1 and α3 isoforms, we establish that the molecular basis of specificity resides in the differential positioning of the armadillo repeats 7 and 8. Overall, our study provides mechanistic insights into a range of important nucleocytoplasmic transport processes reliant on isoform adaptor specificity.

Towards Novel Herbicide Modes of Action by Inhibiting Lysine Biosynthesis in Plants

Publisher: Cold Spring Harbor Laboratory

Date: 06-05-2021

DOI: 10.1101/2021.05.06.442928

Abstract: Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. Using a high throughput chemical screen, we identified the first class of plant DHDPS inhibitors that have micromolar potency against Arabidopsis thaliana DHDPS isoforms. Employing X-ray crystallography, we determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. We also demonstrated that the inhibitors attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide resistant weeds.

Metal‐Free Synthesis of Functional 1‐Substituted‐1,2,3‐Triazoles from Ethenesulfonyl Fluoride and Organic Azides

Publisher: Wiley

Date: 28-11-2020

DOI: 10.1002/ANIE.201912728

Abstract: The boom in growth of 1,4‐disubstituted triazole products, in particular, since the early 2000’s, can be largely attributed to the birth of click chemistry and the discovery of the Cu I ‐catalyzed azide–alkyne cycloaddition (CuAAC). Yet the synthesis of relatively simple, albeit important, 1‐substituted‐1,2,3‐triazoles has been surprisingly more challenging. Reported here is a straightforward and scalable click‐inspired protocol for the synthesis of 1‐substituted‐1,2,3‐triazoles from organic azides and the bench stable acetylene surrogate ethenesulfonyl fluoride (ESF). The new transformation tolerates a wide selection of substrates and proceeds smoothly under metal‐free conditions to give the products in excellent yield. Under controlled acidic conditions, the 1‐substituted‐1,2,3‐triazole products undergo a Michael addition reaction with a second equivalent of ESF to give the unprecedented 1‐substituted triazolium sulfonyl fluoride salts.

Lysine: biosynthesis, catabolism and roles

Publisher: WikiJournal User Group

Date: 06-2018

DOI: 10.15347/WJS/2018.004

Defining the distinct, intrinsic properties of the novel type I interferon, epsilon

Publisher: Elsevier BV

Date: 03-2017

DOI: 10.1074/JBC.M117.800755

Estimating the Effectiveness of Perinatal Care Technologies by Expert Opinion

Publisher: Cambridge University Press (CUP)

Date: 1991

DOI: 10.1017/S0266462300005742

Abstract: The majority of technologies in use in perinatal care were organized into 45 technological functions. Forty-six experts from 19 different regions of Brazil and other Latin American countries then selected a “basic package” (BP) of 15 technological functions. Considering the 12 main causes of perinatal mortality in Brazil, the experts estimated the number of preventable deaths, assuming universal coverage by the BP and the additional reductions that could be obtained by gradually adding other technological functions to the BP. A simulation was performed for the 26 states of Brazil to identify regional priorities for the diffusion of technological functions. For most regions, the BP appears to be the most effective intervention, with the potential of reducing perinatal mortality by 33%, followed by “coordination of services and referral of pregnant women” (14%), and “treatment of respiratory conditions” (11.8%).

Structural insight into the Scribble PDZ domains interaction with the oncogenic Human T‐cell lymphotrophic virus‐1 ( HTLV ‐1) Tax1 PBM

Publisher: Wiley

Date: 06-09-2022

DOI: 10.1111/FEBS.16607

Abstract: Scribble (Scrib) is a highly conserved cell polarity regulator that harbours potent tumour suppressor activity and plays an important role in cell migration. Dysregulation of polarity is associated with poor prognosis during viral infections. Human T‐cell lymphotrophic virus‐1 (HTLV‐1) encodes for the oncogenic Tax1 protein, a modulator of the transcription of viral and human proteins that can cause cell cycle dysregulation as well as a loss of genomic integrity. Previous studies established that Scribble interacts with Tax1 via its C‐terminal PDZ‐binding motif (PBM), leading to aggregation of polarity regulators and subsequent perturbation of host cell adhesion, proliferation, and signalling. Using isothermal titration calorimetry, we now show that all four PDZ domains of Scribble bind to Tax1 PBM. We then determined crystal structures of Scribble PDZ1, PDZ2 and PDZ3 domains bound to Tax1 PBM. Our findings establish a structural basis for Tax1‐mediated subversion of Scribble‐mediated cell polarity signalling and provide the platform for mechanistic studies to examine Tax1 induced mislocalization of Scribble and the associated changes in cellular architecture and subsequent tumorigenesis.

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase

Publisher: Oxford University Press (OUP)

Date: 08-05-2021

DOI: 10.1093/PLCELL/KOAB130

Abstract: Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation ex le known to occur in nature.

Dual roles for a non-conserved amino acid in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus

Publisher: Wiley

Date: 21-11-2014

DOI: 10.1111/MMI.12446

Abstract: Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

Biosynthesis of uridine diphosphate N‐Acetylglucosamine: An underexploited pathway in the search for novel antibiotics?

Publisher: Wiley

Date: 26-07-2022

DOI: 10.1002/IUB.2664

Abstract: Although the prevalence of antibiotic resistance is increasing at an alarming rate, there are a dwindling number of effective antibiotics available. Thus, the development of novel antibacterial agents should be of utmost importance. Peptidoglycan biosynthesis has been and is still an attractive source for antibiotic targets however, there are several components that remain underexploited. In this review, we examine the enzymes involved in the biosynthesis of one such component, UDP‐ N ‐acetylglucosamine, an essential building block and precursor of bacterial peptidoglycan. Furthermore, given the presence of a similar biosynthesis pathway in eukaryotes, we discuss the current knowledge on the differences and similarities between the bacterial and eukaryotic enzymes. Finally, this review also summarises the recent advances made in the development of inhibitors targeting the bacterial enzymes.

Structure and Function of Cyanobacterial DHDPS and DHDPR

Publisher: Springer Science and Business Media LLC

Date: 15-11-2016

DOI: 10.1038/SREP37111

Abstract: Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the ergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis . The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR erged after cyanobacteria evolved.

Optimising in situ click chemistry: The screening and identification of biotin protein ligase inhibitors

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3SC51127H

Shapeshifting bullvalene-linked vancomycin dimers as effective antibiotics against multidrug-resistant gram-positive bacteria

Publisher: Proceedings of the National Academy of Sciences

Date: 03-04-2023

DOI: 10.1073/PNAS.2208737120

Abstract: The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here, we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a triazole-linked bullvalene core, exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding d -Ala- d -Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, implying the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.

Structure guided design of Biotin protein ligase inhibitors for antibiotic discovery

Publisher: Bentham Science Publishers Ltd.

Date: 31-12-2014

DOI: 10.2174/1568026613666131111103149

Abstract: Biotin protein ligase (BPL) represents a promising target for the discovery of new antibacterial chemotherapeutics. Here we review the central role of BPL for the survival and virulence of clinically important Staphylococcus aureus in support of this claim. X-ray crystallography structures of BPLs in complex with ligands and small molecule inhibitors provide new insights into the mechanism of protein biotinylation, and a template for structure guided approaches to the design of inhibitors for antibacterial discovery. Most BPLs employ an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5´-AMP from substrates biotin and ATP. Recent studies reporting chemical analogs of biotin and biotinyl-5´-AMP as BPL inhibitors that represent new classes of anti-S. aureus agents are reviewed. We highlight strategies to selectively inhibit bacterial BPL over the mammalian equivalent using a 1,2,3-triazole isostere to replace the labile phosphoanhydride naturally present in biotinyl-5´-AMP. A novel in situ approach to improve the detection of triazole-based inhibitors is also presented that could potentially be widely applied to other protein targets.

Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms

Publisher: Wiley

Date: 02-03-2021

DOI: 10.1111/FEBS.15766

Abstract: Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate‐limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l ‐lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine‐mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10‐fold more sensitive to lysine. We subsequently used X‐ray crystallography to probe for structural differences between the apo‐ and lysine‐bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6‐fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI .

Heparanase: Cloning, Function and Regulation

Publisher: Springer International Publishing

Date: 2020

DOI: 10.1007/978-3-030-34521-1_7

Structural insight into tanapoxvirus‐mediated inhibition of apoptosis

Publisher: Wiley

Date: 31-05-2020

DOI: 10.1111/FEBS.15365

Abstract: Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double‐stranded DNA virus belonging to the poxviridae that causes mild monkeypox‐like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl‐2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl‐2 fold comprising 7 α‐helices and utilizes the canonical Bcl‐2 binding groove to engage proapoptotic host cell Bcl‐2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain‐swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain‐swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus‐mediated inhibition of host cell apoptosis and reveal the capacity of Bcl‐2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. Structural data are available in the Protein Data Bank (PDB) under the accession numbers 6TPQ , 6TQQ and 6TRR .

A F420-dependent single domain chemogenetic tool for protein de-dimerization

Publisher: Cold Spring Harbor Laboratory

Date: 08-11-2022

DOI: 10.1101/2022.11.07.515376

Abstract: Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F 420 , which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F 420 , MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F 420 binding results in the dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F 420 -dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ .

From bacteria to plants: a repurposing strategy in the pursuit for novel herbicides

Publisher: Cold Spring Harbor Laboratory

Date: 21-02-2022

DOI: 10.1101/2022.02.20.481218

Repurposed inhibitor of bacterial dihydrodipicolinate reductase exhibits effective herbicidal activity

Publisher: Springer Science and Business Media LLC

Date: 22-05-2023

DOI: 10.1038/S42003-023-04895-Y

Abstract: Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicide-resistant weeds. Here, we employed a novel strategy to repurpose a ‘failed’ antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana . We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana . We also showed that our lead compound is the first lysine biosynthesis inhibitor with activity against both monocotyledonous and dicotyledonous weed species, by demonstrating its effectiveness at reducing the germination and growth of Lolium rigidum (rigid ryegrass) and Raphanus raphanistrum (wild radish). These results provide proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing ‘failed’ antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes.

Review: amino acid biosynthesis as a target for herbicide development

Publisher: Wiley

Date: 22-06-2020

DOI: 10.1002/PS.5943

SuFExable Polymers with Helical Structures Derived from Thionyl Tetrafluoride (SOF4)

Publisher: American Chemical Society (ACS)

Date: 23-10-2019

DOI: 10.26434/CHEMRXIV.10005758.V1

Abstract: The first SuFEx click chemistry synthesis of SOF 4 -derived copolymers based upon the polymerization of bis(iminosulfur oxydifluorides) and bis(aryl silyl ethers) is described. This novel class of SuFEx polymer presents two key characteristics: First, the newly created [-N=S(=O)F-O-] polymer backbone linkages are themselves SuFExable and primed to undergo further high-yielding and precise SuFEx-based post-modification with phenols or amines to yield branched functional polymers. Second, studies of in idual polymer chains of several of these new materials indicate the presence of helical polymer structures, which itself suggests a preferential approach of new monomers onto the growing polymer chain upon the formation of the stereogenic linking moiety.

How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase?

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BIOCHI.2010.03.004

Abstract: Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a validated antibiotic target, catalyses the first committed step in the lysine biosynthetic pathway: the condensation reaction between (S)-aspartate beta-semialdehyde [(S)-ASA] and pyruvate via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Escherichia coli DHDPS mutants K161A and K161R of the active-site lysine were characterised for the first time. Unexpectedly, the mutant enzymes were still catalytically active, albeit with a significant decrease in activity. The k(cat) values for DHDPS-K161A and DHDPS-K161R were 0.06 +/- 0.02 s(-1) and 0.16 +/- 0.06 s(-1) respectively, compared to 45 +/- 3 s(-1) for the wild-type enzyme. Remarkably, the K(M) values for pyruvate increased by only 3-fold for DHDPS-K161A and DHDPS-K161R (0.45 +/- 0.04 mM and 0.57 +/- 0.06 mM, compared to 0.15 +/- 0.01 mM for the wild-type DHDPS), while the K(M) values for (S)-ASA remained the same for DHDPS-K161R (0.12 +/- 0.01 mM) and increased by only 2-fold for DHDPS-K161A (0.23 +/- 0.02 mM) and the K(i) for lysine was unchanged. The X-ray crystal structures of DHDPS-K161A and DHDPS-K161R were solved at resolutions of 2.0 and 2.1 A respectively and showed no changes in their secondary or tertiary structures when compared to the wild-type structure. The crystal structure of DHDPS-K161A with pyruvate bound at the active site was solved at a resolution of 2.3 A and revealed a defined binding pocket for pyruvate that is thus not dependent upon lysine 161. Taken together with ITC and NMR data, it is concluded that although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis.

Supervisor types: which one is your match?

Publisher: Springer Science and Business Media LLC

Date: 08-2016

DOI: 10.1038/CDDISCOVERY.2016.60

Synthesis and structure-activity relationship studies of 2,4-thiazolidinediones and analogous heterocycles as inhibitors of dihydrodipicolinate synthase

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.BMC.2021.116518

Abstract: Dihydrodipicolinate synthase (DHDPS), responsible for the first committed step of the diaminopimelate pathway for lysine biosynthesis, has become an attractive target for the development of new antibacterial and herbicidal agents. Herein, we report the discovery and exploration of the first inhibitors of E. coli DHDPS which have been identified from screening lead and are not based on substrates from the lysine biosynthesis pathway. Over 50 thiazolidinediones and related analogues have been prepared in order to thoroughly evaluate the structure-activity relationships against this enzyme of significant interest.

Author response: Towards novel herbicide modes of action by inhibiting lysine biosynthesis in plants

Publisher: eLife Sciences Publications, Ltd

Date: 20-07-2021

DOI: 10.7554/ELIFE.69444.SA2

Metal‐Free Synthesis of Functional 1‐Substituted‐1,2,3‐Triazoles from Ethenesulfonyl Fluoride and Organic Azides

Publisher: Wiley

Date: 28-11-2020

DOI: 10.1002/ANGE.201912728

Abstract: The boom in growth of 1,4‐disubstituted triazole products, in particular, since the early 2000’s, can be largely attributed to the birth of click chemistry and the discovery of the Cu I ‐catalyzed azide–alkyne cycloaddition (CuAAC). Yet the synthesis of relatively simple, albeit important, 1‐substituted‐1,2,3‐triazoles has been surprisingly more challenging. Reported here is a straightforward and scalable click‐inspired protocol for the synthesis of 1‐substituted‐1,2,3‐triazoles from organic azides and the bench stable acetylene surrogate ethenesulfonyl fluoride (ESF). The new transformation tolerates a wide selection of substrates and proceeds smoothly under metal‐free conditions to give the products in excellent yield. Under controlled acidic conditions, the 1‐substituted‐1,2,3‐triazole products undergo a Michael addition reaction with a second equivalent of ESF to give the unprecedented 1‐substituted triazolium sulfonyl fluoride salts.

Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum

Publisher: Springer Science and Business Media LLC

Date: 09-05-2018

DOI: 10.1007/S00425-018-2894-X

Abstract: Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a K

Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1016/J.PEP.2018.01.003

Abstract: Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, K

Structural characterisation of a MAPR-related archaeal cytochrome b

Publisher: Wiley

Date: 22-08-2022

DOI: 10.1002/1873-3468.14471

Abstract: We recently reported that the membrane-associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b

Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation

Publisher: Wiley

Date: 05-08-2020

DOI: 10.1111/FEBS.15014

Abstract: Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital-acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso-DAP and lysine. The rate-limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (k

Author response: A dual-target herbicidal inhibitor of lysine biosynthesis

Publisher: eLife Sciences Publications, Ltd

Date: 08-06-2022

DOI: 10.7554/ELIFE.78235.SA2

Structural insight into tanapoxvirus mediated inhibition of apoptosis

Publisher: Cold Spring Harbor Laboratory

Date: 23-01-2020

DOI: 10.1101/2020.01.21.914671

Abstract: Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double-stranded DNA virus belonging to the poxviridae that causes mild Monkeypox-like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins, and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric as well as a domain-swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain-swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus mediated inhibition of host cell apoptosis, and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state.

Diversity in the intrinsic apoptosis pathway of nematodes

Publisher: Springer Science and Business Media LLC

Date: 28-08-2020

DOI: 10.1038/S42003-020-01208-5

Abstract: Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans . Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans , 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans , or other nematode taxa studied.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4

Publisher: Springer Science and Business Media LLC

Date: 16-07-2019

DOI: 10.1038/S41598-019-46500-5

Abstract: We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum . To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na + ) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na + dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na + inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na + into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na + within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1

Publisher: Informa UK Limited

Date: 09-01-2019

DOI: 10.1080/15548627.2018.1564557

Charles Sturt University - Wagga Wagga Campus

Location: Australia

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University Of Adelaide

Location: Australia

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La Trobe University

Location: Australia

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University Of Canterbury

Location: New Zealand

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Fighting Superbugs With Novel Drugs To Tackle Resistance

Start Date: 2019

End Date: 2022

Funder: Defence Science Institute

Towards Herbicide Cocktails With A New Mode Of Action To Avert Resistance

Start Date: 2019

End Date: 2022

Funder: Australian Research Council

Broad Spectrum Inhibition Of An Enzyme Antibiotic Target

Start Date: 2015

End Date: 2019

Funder: National Health and Medical Research Council

Using Supercomputers In The Search For Herbicides That Inhibit Amino Acid Production In Plants

Start Date: 2022

End Date: 2024

Funder: Australian Academy of Science

Re-purposing Shelved ‘antibiotics’ In The Search For New Herbicides

Start Date: 2022

End Date: 2025

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT230100203

Start Date: 07-2023

End Date: 06-2027

Amount: $830,380.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220101901

Start Date: 06-2022

End Date: 06-2025

Amount: $446,368.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC220100050

Start Date: 07-2023

End Date: 07-2028

Amount: $4,508,426.00

Funder: Australian Research Council

Discovery Early Career Researcher Award - Grant ID: DE190100806

Start Date: 03-2019

End Date: 12-2023

Amount: $419,854.00

Funder: Australian Research Council