Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the ...parasite’s life cycle. We defined the structure–activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted “gatekeeper” residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.
Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe ...the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.
Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence ...of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.
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•The structure of fungal pantothenate kinase (PanK) was solved at 1.8 Å resolution•A high-throughput chemical screen identified pyrimidone triazols as PanK inhibitors•Structure-function analyses revealed essential residues for PanK catalysis•Fungal PanK are valid targets for antifungal drug development
In this work, Gihaz et al. report the crystal structure of a pantothenate kinase (PanK) from Saccharomyces cerevisiae and the discovery of new inhibitors with antifungal activity. Structural, biochemical, and genetic evidence validate fungal PanKs as promising targets for antifungal drug development.
Highlights • New beta-lactamase inhibitor with novel mechanism of action is reviewed. • Methods of targeting bacterial gyrase compounds are discussed. • Structures of proteins from orthologous ...species can be used for drug design and discovery. • Applications of Nuclear Magnetic Resonance to drug discovery are discussed. • Structural genomics programs funded by provide support to independent investigators.
Human topoisomerase I relaxes superhelical tension associated with DNA replication, transcription and recombination by reversibly nicking one strand of duplex DNA and forming a covalent ...3′-phosphotyrosine linkage. This enzyme is the sole target of the camptothecin family of anticancer compounds, which acts by stabilizing the covalent protein–DNA complex and enhancing apoptosis through blocking the advancement of replication forks. Mutations that impart resistance to camptothecin have been identified in several regions of human topoisomerase I. We present the crystal structures of two camptothecin-resistant forms of human topoisomerase I (Phe361Ser at 2.6Å resolution and Asn722Ser at 2.3Å resolution) in ternary complexes with DNA and topotecan (Hycamtin®), a camptothecin analogue currently in widespread clinical use. While the alteration of Asn722 to Ser leads to the elimination of a water-mediated contact between the enzyme and topotecan, we were surprised to find that a well-ordered water molecule replaces the hydrophobic phenylalanine side-chain in the Phe361Ser structure. We further consider camptothecin-resistant mutations at seven additional sites in human topoisomerase I and present structural evidence explaining their possible impact on drug binding. These results advance our understanding of the mechanism of cell poisoning by camptothecin and suggest specific modifications to the drug that may improve efficacy.
The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein ...membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.
The agglutinin-like sequence (Als) proteins are best-characterized in
and known for their role in adhesion of the fungal cell to host and abiotic surfaces.
sequences are often misassembled in ...whole-genome sequence data because each species has multiple
loci that contain similar sequences, most notably tandem copies of highly conserved repeated sequences. The
species complex includes
,
, and
, three distinct but closely related species. Using publicly available genome resources,
genome assemblies, and laboratory experimentation including Sanger sequencing, five
genes were characterized in
strain CDC317, three in
strain 90-125, and four in
strain ATCC 96143. The newly characterized
genes shared similar features with the well-known
family, but also displayed unique attributes such as novel short, imperfect repeat sequences that were found in other genes encoding fungal cell-wall proteins. Evidence of recombination between
sequences and other genes was most obvious in
, which had the 5' end of an
gene and the repeated sequences and 3' end from the
family. Together, these results blur the boundaries between the fungal cell-wall families that were defined in
. TaqMan assays were used to quantify relative expression for each
gene. Some measurements were complicated by the assay location within the
gene. Considerable variation was noted in relative gene expression for isolates of the same species. Overall, however, there was a trend toward higher relative gene expression in saturated cultures rather than younger cultures. This work provides a complete description of the
genes in the
species complex and a toolkit that promotes further investigations into the role of the Als proteins in host-fungal interactions.
Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment ...options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N . fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC 50 = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC 50 = 0.21 ± 0.02 μM) while the reported CC 50 was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of –8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.
Fungal infections affect over one billion people worldwide each year and are the leading cause of mortality by eukaryotic pathogens with an estimated 150 million severe life‐threatening cases and 1.7 ...million deaths reported annually. The rapid increase in the number of clinical cases and the emergence of multidrug resistant pathogens such as Candida auris emphasize the urgent need for new strategies to develop more effective, selective and safe antifungals. Coenzyme A (CoA) plays a central role in key cellular processes including the citric acid cycle, lipid metabolism, and ergosterol biosynthesis. The first step in the CoA biosynthesis pathway is catalyzed by a pantothenate kinase (PanK), which phosphorylates pantothenic acid (PA, or vitamin B5) to form phosphopantothenate. In fungi, this enzyme is encoded by a single copy PanK gene, which plays an essential role in cell viability. Here, we report the first crystal structure of the S. cerevisiae PanK enzyme, Cab1, at 1.8Å resolution. Cab1 was crystallized alone and in the presence of three lead high‐affinity inhibitors identified in a high‐throughput chemical screen of a library of 156,593 small molecules. Structure‐guided mutagenesis together with functional complementation assays in yeast identified key residues in Cab1 that are essential for catalysis, substrate binding and dimerization. Structure activity relationship and biological assays identified a lead inhibitor YU385599, with excellent activity against the human pathogens Candida albicans, C. glabrata, and C. parapsilosis. Together, our data validate PanK activity as a novel drug target and identify a novel class of small molecules for the treatment of fungal diseases.
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