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ItemReaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategyXie, SC ; Metcalfe, RD ; Dunn, E ; Morton, CJ ; Huang, S-C ; Puhalovich, T ; Du, Y ; Wittlin, S ; Nie, S ; Luth, MR ; Ma, L ; Kim, M-S ; Pasaje, CFA ; Kumpornsin, K ; Giannangelo, C ; Houghton, FJ ; Churchyard, A ; Famodimu, MT ; Barry, DC ; Gillett, DL ; Dey, S ; Kosasih, CC ; Newman, W ; Niles, JC ; Lee, MCS ; Baum, J ; Ottilie, S ; Winzeler, EA ; Creek, DJ ; Williamson, N ; Parker, MW ; Brand, S ; Langston, SP ; Dick, LR ; Griffin, MDW ; Gould, AE ; Tilley, L (AMER ASSOC ADVANCEMENT SCIENCE, 2022-06-03)Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
ItemThe structure of the extracellular domains of human interleukin 11? receptor reveals mechanisms of cytokine engagementMetcalfe, RD ; Aizel, K ; Zlatic, CO ; Nguyen, PM ; Morton, CJ ; Lio, DS-S ; Cheng, H-C ; Dobson, RCJ ; Parker, MW ; Gooley, PR ; Putoczki, TL ; Griffin, MDW (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2020-06-12)Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11-IL-11Rα complex.
ItemDesign of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasomeXie, SC ; Metcalfe, RD ; Mizutani, H ; Puhalovich, T ; Hanssen, E ; Morton, CJ ; Du, Y ; Dogovski, C ; Huang, S-C ; Ciavarri, J ; Hales, P ; Griffin, RJ ; Cohen, LH ; Chuang, B-C ; Wittlin, S ; Deni, I ; Yeo, T ; Ward, KE ; Barry, DC ; Liu, B ; Gillett, DL ; Crespo-Fernandez, BF ; Ottilie, S ; Mittal, N ; Churchyard, A ; Ferguson, D ; Aguiar, ACC ; Guido, RVC ; Baum, J ; Hanson, KK ; Winzeler, EA ; Gamo, F-J ; Fidock, DA ; Baud, D ; Parker, MW ; Brand, S ; Dick, LR ; Griffin, MDW ; Gould, AE ; Tilley, L (NATL ACAD SCIENCES, 2021-09-28)The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
ItemA Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related ProteinsEvans, JC ; Johnstone, BA ; Lawrence, SL ; Morton, CJ ; Christie, MP ; Parker, MW ; Tweten, RK ; McClane, BA (AMER SOC MICROBIOLOGY, 2020-09-01)The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.