Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase

Rab geranylgeranyltransferase (GGTase-II, RGGT) catalyses the post-translational modification of eukaryotic Rab GTPases, proteins implicated in several pathologies, including cancer, diabetes, neurodegenerative, and infectious diseases. Thus, RGGT inhibitors are believed to be a potential platform for the development of drugs and tools for studying processes related to the abnormal activity of Rab GTPases. Here, a series of new alpha-phosphonocarboxylates have been prepared in the first attempt of rational design of covalent inhibitors of RGGT derived from non-covalent inhibitors. These compounds were equipped with electrophilic groups capable of binding cysteines, which are present in the catalytic cavity of RGGT. A few of these analogues have shown micromolar activity against RGGT, which correlated with their ability to inhibit the proliferation of the HeLa cancer cell line. The proposed mechanism of this inhibitory activity was rationalised by molecular docking and mass spectrometric measurements, supported by stability and reactivity studies

Proteasome activity contributes to prosurvival response upon mild mitochondrial stress in Caenorhabditis elegans

Defects in mitochondrial function activate compensatory responses in the cell. Mitochondrial stress that is caused by unfolded proteins inside the organelle induces a transcriptional response (termed the “mitochondrial unfolded protein response” [UPRmt]) that is mediated by activating transcription factor associated with stress 1 (ATFS-1). The UPRmt increases mitochondrial protein quality control. Mitochondrial dysfunction frequently causes defects in the import of proteins, resulting in the accumulation of mitochondrial proteins outside the organelle. In yeast, cells respond to mistargeted mitochondrial proteins by increasing activity of the proteasome in the cytosol (termed the “unfolded protein response activated by mistargeting of proteins” [UPRam]). The presence and relevance of this response in higher eukaryotes is unclear. Here, we demonstrate that defects in mitochondrial protein import in Caenorhabditis elegans lead to proteasome activation and life span extension. Both proteasome activation and life span prolongation partially depend on ATFS-1, despite its lack of influence on proteasomal gene transcription. Importantly, life span prolongation depends on the fully assembled proteasome. Our data provide a link between mitochondrial dysfunction and proteasomal activity and demonstrate its direct relevance to mechanisms that promote longevity

Global Analysis of Protein N-Myristoylation and Exploration of N-Myristoyltransferase as a Drug Target in the Neglected Human Pathogen Leishmania donovani

N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis

Fig Nuclear accumulation of cellular proteins synthesised prior to infection.

<p>Uninfected Vero cells were pre-labeled with HPG (30 min pulse) followed by an infection with HSV-1 at an MOI of 10 for 4 hr (HSV-1) or mock infection (Mock). Cells were then fixed and stained for Hsc70, followed by click reactions. Cells containing Hsc70 foci were identified and the subnuclear localisation of newly synthesised proteins (green) and Hsc70 (red) are shown with diagonal arrowheads indicating the colocalisation of nuclear NPDs and Hsc70 foci as discussed in the text.</p

Biochemical analysis of newly synthesised proteins using HPG and click chemistry.

<p>(A) Schematic diagram illustrating comparative structures of methionine and HPG. The scheme indicates the in vivo incorporation of HPG into protein (solid black dots within a protein chain) and then the subsequent in vitro cycloaddition reaction to covalently cross link an azide fluorochrome-coupled capture reagent (coloured star) to HPG. (B) Mock-infected Vero cells were pulse-labeled using 1 mM HPG for 1 hr, lysed and subjected to click reactions using IRDye 800CW Azide Infrared Dye. Proteins were separated by SDS-PAGE and visualised by in-gel fluorescence using a LI-COR Odyssey Infrared Imaging System. Control experiments were carried out either in the absence of HPG (lane 5) or in the presence of 100 μg/ml of CHX (lane 4). Lanes 1–3 represent the total Coomassie blue staining protein profile and lanes 4–6 the in-gel fluorescence profile of the identical gel. (C) Cell viability of uninfected Vero cells (% live cells, in triplicate) was assessed by trypan blue exclusion. Control cultures were subject to either no methionine depletion and incubation in standard methionine-containing medium (Con; white bar) or methionine depletion with subsequent incubation in standard methionine-containing medium (Met; light grey bar); while HPG labelling was performed after methionine depletion with subsequent incubation in HPG -containing medium (30 min pulse). (D) In-gel fluorescence of newly synthesised proteins in total, cytosolic and nuclear fractions. Mock or HSV infected Vero cells (MOI 10) were pulse-labeled at the times indicated for 1 hr, lysed and fractionated prior to click reaction. Equal concentrations of proteins (20 μg, representing a 4-fold increased loading by cell equivalents for the nuclear fraction) were resolved by SDS-PAGE, and proteins visualised using a LI-COR Odyssey Infrared Imaging System scanned into the green channel. (E) The same gel was stained with Coomassie brilliant blue for total protein detection. Representative host cell proteins enriched within the cytoplasmic and nuclear fractions are labeled HC and HN respectively. (F) The same samples after separation by SDS-PAGE where transferred to a nitrocellulose membrane. Total steady-state levels of candidate viral proteins, ICP4, ICP0 and VP5 (red) were simultaneously detected using monoclonal antibodies, and newly synthesised proteins (green) were visualised on the blot.</p

ICP22 localises to NPDs and phase-dense nuclear bodies.

<p>Infected Vero cells were either untreated (standard media; control) or HPG pulse-labeled for 30 min at 2 hr p.i. (HPG), fixed and analysed by fluorescence (for newly synthesised proteins (green) and ICP22 (red) and by phase microscopy. Diagonal arrowheads indicate the colocalisation of NPDs with ICP22 as well as phase-dense nuclear domains. The inset shows the precise co-localisation of ICP22 with NPDs. The punctate localisation of ICP22, and its recruitment into phase dense bodies was independent of HPG pulse-labeling and also observed in the control infected cultures in the absence of HPG(Control).</p