Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various ...molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Targeted delivery of potent cytotoxic drugs to cancer cells minimizes systemic toxicity and several side effects. NHC*−Au−Cl has already been proven to be a potent anticancer agent. In this study, we ...explore a strategy based on chemoselective cysteine conjugation of NHC*−Au−Cl to albumin and trastuzumab (Thiomab LC‐V205C) to potentiate drug‐ligand ratio, pharmacokinetics, as well as drug efficacy and safety. This strategy is a step forward towards the use of gold‐based anticancer agents as targeted therapies.
Golden therapy: Synthesis of NHC*−Au−S‐protein bioconjugates to increase stability, modulate pharmacokinetics, and ultimately enhance efficacy and safety of gold‐based anticancer drugs. The findings reported represent a step forward towards the use of gold‐based anticancer drugs as targeted therapies.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The malaria‐causing parasite, Plasmodium, contains a unique non‐photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane ...transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast‐localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast‐localised. Interestingly, three apicoplast‐localised candidates lack a canonical apicoplast targeting signal but do contain conserved N‐terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Salmon calcitonin (sCT) was conjugated
via its
N-terminal cysteine to a comb-shaped end-functionalized poly(poly(ethylene glycol) methyl ether methacrylate) (PolyPEG®, 6.5 kDa), and to linear PEG ...(5 kDa). Conjugate molecular weight and purity was assessed by SEC–HPLC and MALDI–TOF MS. Bioactivity of conjugates was measured by cyclic AMP assay in T47D cells. Calcium and calcitonin levels were measured in rats following intravenous injections. Stability of conjugates was tested against serine proteases, intestinal and liver homogenates and serum. Cytotoxicity of conjugates was assessed by lactate dehydrogenase (LDH) assay and by haemolytic assay of rat red blood cells. Results showed that the two conjugates were of high purity with molecular weights similar to predictions. Both conjugates retained more than 85% bioactivity
in
vitro and had nanomolar EC
50 values similar to sCT. While both sCT-PolyPEG®
6.5 K and sCT-PEG
5 K were resistant to metabolism by serine proteases, homogenates and serum, PolyPEG®
6.5 K was more so. Although both conjugates reduced serum calcium to levels similar to those achieved with sCT, PolyPEG®
6.5 K extended the
T
1/2 and AUC of serum sCT over values achieved with sCT-PEG and sCT itself. None of PolyPEG®, PEG or methacrylic acid displayed significant cytotoxicity. PolyPEG® may therefore have potential to improve pharmacokinetic profiles of injected peptides.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel comb-shaped end-functionalised (poly(PEG) methyl ether methacrylate) (sCT-P) polymers, to yield conjugates of total molecular weights ...(MW) inclusive of sCT: 6.5, 9.5, 23 and 40
kDa. The conjugates were characterised by HPLC and their
in vitro and
in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of E
max and an EC
50 of 3.7
nM.
In vivo, while free sCT and the conjugates reduced serum calcium to a maximum of 15–30% over 240
min, the half-life (T
1/2) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40
kDa). Mathematical modelling accurately predicted the MW relationships to
in vitro efficacy, potency,
in vivo PK and enzymatic stability. With a significant increase in T
1/2 for sCT, the 40
kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation.
Concentration–response curves of salmon calcitonin (sCT) and comb-shaped PEGylated-sCT conjugates of increasing molecular weight in an
in vitro cAMP bioassay. sCT □, sCT-P (6.5
kDa) ◊, sCT-P (9.5
kDa) ■, sCT-P (23
kDa) ○, and sCT-P (40
kDa) ♦.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is ...required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.
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•A genome-scale screen identifies genes for Plasmodium berghei sexual development•scRNA-seq phenotyping places mutants in the context of a transcriptomic atlas•A LOTUS/OST-HTH domain gene is a conserved factor determining male sex•Protein interactors point to posttranscriptional mechanisms for sex determination
Russell et al. systematically reveal early sexual development genes that a malaria parasite requires to infect its mosquito vector. Single-cell phenotyping and protein interactions distinguish between functions in sex determination and development and generate initial insights into mechanisms of how male and female sex are determined in a divergent eukaryote.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Glycopolymers have been synthesised by post‐functionalisation of well‐defined alkyne‐functional polymers with sugar azides to yield N‐glycosyl 1,2,3‐triazole functional polymers. The Cu(I)‐catalysed ...Huisgen cycloaddition was used to attach α‐mannoside, β‐galactoside and β‐lactoside derivatives via an azide functionality bound directly to the sugar anomeric carbon. Three different catalytic systems were investigated for the click reactions; (PPh3)3Cu(I)Br, TBTA/Cu(I)Br and bathophenanthrolinedisulphonic acid disodium salt/Cu(I)Br. The latter of these was found to be the most efficient for the attachment of the larger/more sterically hindered disaccharide lactose moiety. The interaction of the lactose‐ and galactose‐bearing glycopolymers with Ricinus Communis Agglutinin (RCA I) lectin was investigated by affinity HPLC analysis. The rate of the interaction between mannose polymer and concanavalin A (Con A) lectin was assessed by turbidimetry. The results from the lectin conjugation studies indicate that the glycopolymers prepared in this work are able to function as multivalent ligands, further suggesting that the attachment of the triazole directly to the sugar anomeric carbon has no significant effect on the interaction of these glycopolymers with Con A and RCA I.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The reaction of salmon calcitonin (sCT) with various linear PEG and PolyPEGs was investigated under a variety of reaction conditions. The optimal experimental conditions for the production of high ...yield monoconjugated sCT were determined using RP-HPLC, SEC-HPLC, IE-FPLC and MALDI-TOF MS analysis. Semi-preparative IE-FPLC was used to purify high yield mono conjugated sCT-5 kDa linear PEG, sCT -6.5 kDa PolyPEG, sCT-20 kDa PolyPEG and sCT-40 kDa PolyPEG coupled to the N-terminal primary amine. The products were analysed using RP-HPLC, SEC-HPLC, SDS-PAGE and the site of attachment was determined by Tryptic mapping combined with LCMS, RP-HPLC and MALDI-TOF MS. Biological activity was measured invitro, sCT derivates were incubated with T47D cells and an ELISA assay was used to measure the amount of cAMP released. The purified conjugates retained a high degree of biological activity compared to the unmodified peptide. Incubation of sCT and sCTconjugates with proteolytic enzymes showed that for sCT-5 kDa linear PEG and 6.5kDa PolyPEG a high degree (60-70 % of the activity at time zero) of biological activity remained after 30 minutes compared to 0% activity for native sCT.
The reaction of salmon calcitonin (sCT) with various linear PEG and PolyPEGs was investigated under a variety of reaction conditions. The optimal experimental conditions for the production of high ...yield monoconjugated sCT were determined using RP-HPLC, SEC-HPLC, IE-FPLC and MALDI-TOF MS analysis. Semi-preparative IE-FPLC was used to purify high yield mono conjugated sCT-5 kDa linear PEG, sCT -6.5 kDa PolyPEG, sCT-20 kDa PolyPEG and sCT-40 kDa PolyPEG coupled to the N-terminal primary amine. The products were analysed using RP-HPLC, SEC-HPLC, SDS-PAGE and the site of attachment was determined by Tryptic mapping combined with LCMS, RP-HPLC and MALDI-TOF MS. Biological activity was measured invitro, sCT derivates were incubated with T47D cells and an ELISA assay was used to measure the amount of cAMP released. The purified conjugates retained a high degree of biological activity compared to the unmodified peptide. Incubation of sCT and sCTconjugates with proteolytic enzymes showed that for sCT-5 kDa linear PEG and 6.5kDa PolyPEG a high degree (60-70 % of the activity at time zero) of biological activity remained after 30 minutes compared to 0% activity for native sCT.
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