I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.
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Feb 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum, which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate, L-Glutamate (L-Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (PfGluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of PfGluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the PfGluRS showed binding of the enzyme substrates, L-Glu, ATP, and L-Glu plus ATP. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the PfGluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs.
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Nov 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Bicyclic azetidine compounds possess antimalarial activity via targeting of the cytoplasmic Plasmodium falciparum (Pf) protein translation enzyme phenylalanine-tRNA synthetase (cFRS). These drugs kill parasites both in vitro and in vivo, including the blood, liver, and transmission developmental stages. Here we present the co-crystal structure of PfcFRS with a potent inhibitor, the bicyclic azetidine BRD7929. Our studies reveal high-affinity binding of BRD7929 with PfcFRS along with exquisite specificity compared with the human enzyme, leading in turn to potent and selective inhibition of the parasite enzyme. Our co-crystal structure shows that BRD7929 binds in the active site in the α subunit of PfcFRS, where it occupies the amino acid site, an auxiliary site, and partially the ATP site. This structural snapshot of inhibitor-bound PfcFRS thus provides a platform for the structure-guided optimization of novel antimalarial compounds.
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Apr 2022
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I24-Microfocus Macromolecular Crystallography
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Manmohan
Sharma
,
Nipun
Malhotra
,
Manickam
Yogavel
,
Karl
Harlos
,
Bruno
Melillo
,
Eamon
Comer
,
Arthur
Gonse
,
Suhel
Parvez
,
Branko
Mitasev
,
Francis G.
Fang
,
Stuart L.
Schreiber
,
Amit
Sharma
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
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Jan 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Abstract: Scaffold modules known as aminoacyl-tRNA synthetase (aaRS)-interacting multifunctional proteins (AIMPs), such as AIMP1/p43, AIMP2/p38 and AIMP3/p18, are important in driving the assembly of multi-aaRS (MARS) complexes in eukaryotes. Often, AIMPs contain an N-terminal glutathione S-transferase (GST)-like domain and a C-terminal OB-fold tRNA-binding domain. Recently, the apicomplexan-specific Plasmodium falciparum p43 protein (Pfp43) has been annotated as an AIMP and its tRNA binding, tRNA import and membrane association have been characterized. The crystal structures of both the N- and C-terminal domains of the Plasmodium vivax p43 protein (Pvp43), which is an ortholog of Pfp43, have been resolved. Analyses reveal the overall oligomeric structure of Pvp43 and highlight several notable features that show Pvp43 to be a soluble, cytosolic protein. The dimeric assembly of the N-terminal GST-like domain of Pvp43 differs significantly from canonical GST dimers, and it is tied to the C-terminal tRNA-binding domain via a linker region. This work therefore establishes a framework for dissecting the additional roles of p43 orthologs in eukaryotic multi-protein MARS complexes.
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Feb 2020
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I24-Microfocus Macromolecular Crystallography
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Beatriz
Baragaña
,
Barbara
Forte
,
Ryan
Choi
,
Stephen
Nakazawa Hewitt
,
Juan A.
Bueren-Calabuig
,
Joao Pedro
Pisco
,
Caroline
Peet
,
David M.
Dranow
,
David A.
Robinson
,
Chimed
Jansen
,
Neil R.
Norcross
,
Sumiti
Vinayak
,
Mark
Anderson
,
Carrie F.
Brooks
,
Caitlin A.
Cooper
,
Sebastian
Damerow
,
Michael
Delves
,
Karen
Dowers
,
James
Duffy
,
Thomas E.
Edwards
,
Irene
Hallyburton
,
Benjamin G.
Horst
,
Matthew A.
Hulverson
,
Liam
Ferguson
,
María Belén
Jiménez-Díaz
,
Rajiv S.
Jumani
,
Donald D.
Lorimer
,
Melissa S.
Love
,
Steven
Maher
,
Holly
Matthews
,
Case W.
Mcnamara
,
Peter
Miller
,
Sandra
O’neill
,
Kayode K.
Ojo
,
Maria
Osuna-Cabello
,
Erika
Pinto
,
John
Post
,
Jennifer
Riley
,
Matthias
Rottmann
,
Laura M.
Sanz
,
Paul
Scullion
,
Arvind
Sharma
,
Sharon M.
Shepherd
,
Yoko
Shishikura
,
Frederick R. C.
Simeons
,
Erin E.
Stebbins
,
Laste
Stojanovski
,
Ursula
Straschil
,
Fabio K.
Tamaki
,
Jevgenia
Tamjar
,
Leah S.
Torrie
,
Amélie
Vantaux
,
Benoît
Witkowski
,
Sergio
Wittlin
,
Manickam
Yogavel
,
Fabio
Zuccotto
,
Iñigo
Angulo-Barturen
,
Robert
Sinden
,
Jake
Baum
,
Francisco-Javier
Gamo
,
Pascal
Mäser
,
Dennis E.
Kyle
,
Elizabeth A.
Winzeler
,
Peter J.
Myler
,
Paul G.
Wyatt
,
David
Floyd
,
David
Matthews
,
Amit
Sharma
,
Boris
Striepen
,
Christopher D.
Huston
,
David W.
Gray
,
Alan H.
Fairlamb
,
Andrei V.
Pisliakov
,
Chris
Walpole
,
Kevin D.
Read
,
Wesley C.
Van Voorhis
,
Ian H.
Gilbert
Diamond Proposal Number(s):
[10071]
Open Access
Abstract: Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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Apr 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by rise and spread of drug resistance mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX’s effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.
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Aug 2018
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I03-Macromolecular Crystallography
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Pronay
Das
,
Palak
Babbar
,
Nipun
Malhotra
,
Manmohan
Sharma
,
Gorakhnath R.
Jachak
,
Rajesh G.
Gonnade
,
Dhanasekaran
Shanmugam
,
Karl
Harlos
,
Manickam
Yogavel
,
Amit
Sharma
,
D. Srinivasa
Reddy
Diamond Proposal Number(s):
[14744]
Abstract: The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral centre anti-malarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme- and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency where changes at C3 are sensed by rotameric flipping of Glutamate332. Given that scores of anti-malarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of anti-microbial drug development.
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May 2018
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I04-Macromolecular Crystallography
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Vitul
Jain
,
Manickam
Yogavel
,
Haruhisa
Kikuchi
,
Yoshiteru
Oshima
,
Norimitsu
Hariguchi
,
Makoto
Matsumoto
,
Preeti
Goel
,
Bastien
Touquet
,
Rajiv S.
Jumani
,
Fabienne
Tacchini-Cottier
,
Karl
Harlos
,
Christopher D.
Huston
,
Mohamed-Ali
Hakimi
,
Amit
Sharma
Diamond Proposal Number(s):
[14744]
Abstract: Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
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Aug 2017
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