An Essential Farnesylated Kinesin in Trypanosoma brucei

Kinesins are a family of motor proteins conserved throughout eukaryotes. In our present study we characterize a novel kinesin, KinesinCaaX, orthologs of which are only found in the kinetoplastids and not other eukaryotes. KinesinCaaX has the CVIM amino acids at the C-terminus, and CVIM was previously shown to be an ideal signal for protein farnesylation in T. brucei. In this study we show KinesinCaaX is farnesylated using radiolabeling studies and that farnesylation is dependent on the CVIM motif. Using RNA interference, we show KinesinCaaX is essential for T. brucei proliferation. Additionally RNAi KinesinCaaX depleted T. brucei are 4 fold more sensitive to the protein farneysltransferase (PFT) inhibitor LN-59, suggesting that KinesinCaaX is a target of PFT inhibitors' action to block proliferation of T. brucei. Using tetracycline-induced exogenous tagged KinesinCaaX and KinesinCVIMdeletion (non-farnesylated Kinesin) expression lines in T. brucei, we demonstrate KinesinCaaX is farnesylated in T. brucei cells and this farnesylation has functional effects. In cells expressing a CaaX-deleted version of Kinesin, the localization is more diffuse which suggests correct localization depends on farnesylation. Through our investigation of cell cycle, nucleus and kinetoplast quantitation and immunofluorescence assays an important role is suggested for KinesinCaaX in the separation of nuclei and kinetoplasts during and after they have been replicated. Taken together, our work suggests KinesinCaaX is a target of PFT inhibition of T. brucei cell proliferation and KinesinCaaX functions through both the motor and farnesyl groups

Bioactivity of Farnesyltransferase Inhibitors Against Entamoeba histolytica and Schistosoma mansoni

The protozoan parasite Entamoeba histolytica can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the Schistosoma flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both E. histolytica and Schistosoma mansoni genomes encode FT genes. In this study, we phenotypically screened E. histolytica and S. mansoni in vitro with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of Trypanosoma brucei and Trypanosoma cruzi. For E. histolytica, we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 μM against E. histolytica. Apart from five analogs of tipifarnib exhibiting activity against both E. histolytica and S. mansoni, 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 μM after 24 h). Analysis of the structure-activity relationship available for the T. brucei FT suggests that FT may not be the relevant target in E. histolytica and S. mansoni. For E. histolytica, combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases

Setting our sights on infectious diseases

In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings

Isospin symmetry in B(E2) values: Coulomb excitation study of Mg-21

The $T_z$~=~$-\frac{3}{2}$ nucleus ${}^{21}$Mg has been studied by Coulomb excitation on ${}^{196}$Pt and ${}^{110}$Pd targets. A 205.6(1)-keV $\gamma$-ray transition resulting from the Coulomb excitation of the $\frac{5}{2}^+$ ground state to the first excited $\frac{1}{2}^+$ state in ${}^{21}$Mg was observed for the first time. Coulomb excitation cross-section measurements with both targets and a measurement of the half-life of the $\frac{1}{2}^+$ state yield an adopted value of $B(E2;\frac{5}{2}^+\rightarrow\frac{1}{2}^+)$~=~13.3(4)~W.u. A new excited state at 1672(1)~keV with tentative $\frac{9}{2}^+$ assignment was also identified in ${}^{21}$Mg. This work demonstrates large difference of the $B(E2;\frac{5}{2}^+\rightarrow\frac{1}{2}^+)$ values between $T$~=~$\frac{3}{2}$, $A$~=~21 mirror nuclei. The difference is investigated in the shell-model framework employing both isospin conserving and breaking USD interactions and using modern \textsl{ab initio} nuclear structure calculations, which have recently become applicable in the $sd$ shell.Comment: 8 pages, 6 figures, submitted to Phys. Rev. C, Rapid Communicatio

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery