Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis.

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic 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 that 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 value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC 50 value of 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 conclusion of the experiment, 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. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

Characterization of a family I inorganic pyrophosphatase from Legionella pneumophila Philadelphia 1.

Inorganic pyrophosphate (PPi) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PPi must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PPi into two orthophosphates (Pi), preventing the toxic accumulation of the PPi byproduct in cells and making Pi available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Šresolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) ß-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.

Bacterial structural genomics target enabled by a recently discovered potent fungal acetyl-CoA synthetase inhibitor.

The compound ethyl-adenosyl monophosphate ester (ethyl-AMP) has been shown to effectively inhibit acetyl-CoA synthetase (ACS) enzymes and to facilitate the crystallization of fungal ACS enzymes in various contexts. In this study, the addition of ethyl-AMP to a bacterial ACS from Legionella pneumophila resulted in the determination of a co-crystal structure of this previously elusive structural genomics target. The dual functionality of ethyl-AMP in both inhibiting ACS enzymes and promoting crystallization establishes its significance as a valuable resource for advancing structural investigations of this class of proteins.

Structural characterisation of hemagglutinin from seven Influenza A H1N1 strains reveal diversity in the C05 antibody recognition site.

Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines.

In Vitro and In Vivo Efficacy of NITD-916 against Mycobacterium fortuitum.

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.

Discovery and Characterization of Potent, Efficacious, Orally Available Antimalarial Plasmepsin X Inhibitors and Preclinical Safety Assessment of UCB7362.

Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9 log 10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.

Efficacy and Mode of Action of a Direct Inhibitor of Mycobacterium abscessus InhA.

There is an unmet medical need for effective treatments against Mycobacterium abscessus pulmonary infections, to which cystic fibrosis (CF) patients are particularly vulnerable. Recent studies showed that the antitubercular drug isoniazid is inactive against M. abscessus due to the incapacity of the catalase-peroxidase to convert the pro-drug into a reactive metabolite that inhibits the enoyl-ACP reductase InhA. To validate InhAMAB as a druggable target in M. abscessus, we assayed the activity of NITD-916, a 4-hydroxy-2-pyridone lead candidate initially described as a direct inhibitor of InhA that bypasses KatG bioactivation in Mycobacterium tuberculosis. The compound displayed low MIC values against rough and smooth clinical isolates in vitro and significantly reduced the bacterial burden inside human macrophages. Moreover, treatment with NITD-916 reduced the number and size of intracellular mycobacterial cords, regarded as markers of the severity of the infection. Importantly, NITD-916 significantly lowered the M. abscessus burden in CF-derived lung airway organoids. From a mechanistic perspective, NITD-916 abrogated de novo synthesis of mycolic acids and NITD-916-resistant spontaneous mutants harbored point mutations in InhAMAB at residue 96. That NITD-916 targets InhAMAB directly without activation requirements was confirmed genetically and by resolving the crystal structure of the protein in complex with NADH and NITD-916. These findings collectively indicate that InhAMAB is an attractive target to be exploited for future chemotherapeutic developments against this difficult-to-treat mycobacterium and highlight the potential of NITD-916 derivatives for further evaluation in preclinical settings.

Regulation of multiple dimeric states of E-cadherin by adhesion activating antibodies revealed through Cryo-EM and X-ray crystallography.

E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-electron microscopy (EM) and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E-cadherin monomers, but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.

Discovery and Characterization of a Novel Series of Chloropyrimidines as Covalent Inhibitors of the Kinase MSK1.

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.

Characterization of Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Expression and structure of the Chlamydia trachomatis DksA ortholog.

Chlamydia trachomatis is a bacterial obligate intracellular parasite and a significant cause of human disease, including sexually transmitted infections and trachoma. The bacterial RNA polymerase-binding protein DksA is a transcription factor integral to the multicomponent bacterial stress response pathway known as the stringent response. The genome of C. trachomatis encodes a DksA ortholog (DksACt) that is maximally expressed at 15-20 h post infection, a time frame correlating with the onset of transition between the replicative reticulate body (RB) and infectious elementary body (EB) forms of the pathogen. Ectopic overexpression of DksACt in C. trachomatis prior to RB-EB transitions during infection of HeLa cells resulted in a 39.3% reduction in overall replication (yield) and a 49.6% reduction in recovered EBs. While the overall domain organization of DksACt is similar to the DksA ortholog of Escherichia coli (DksAEc), DksACt did not functionally complement DksAEc. Transcription of dksACt is regulated by tandem promoters, one of which also controls expression of nrdR, encoding a negative regulator of deoxyribonucleotide biosynthesis. The phenotype resulting from ectopic expression of DksACt and the correlation between dksACt and nrdR expression is consistent with a role for DksACt in the C. trachomatis developmental cycle.

Structural Characterization of Cytochrome c'ß-Met from an Ammonia-Oxidizing Bacterium.

The ammonia-oxidizing bacterium Nitrosomonas europaea expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NH2OH) is converted to nitric oxide (NO) and nitrous oxide (N2O) requiring a unique heme-lysyl cross-link in the catalytic cofactor. In the second, cyt c'ß-Met, the cross-link is absent, and the cytochrome instead binds H2O2 forming a ferryl species similar to compound II of peroxidases. Here, we report the 1.80 Å crystal structure of cyt c'ß-Met─a well-expressed protein in N. europaea with a lysine to a methionine replacement at the cross-linking position. The structure of cyt c'ß-Met is characterized by a large ß-sheet typical of P460 members; however, several localized structural differences render cyt c'ß-Met distinct. This includes a large lasso-like loop at the "top" of the cytochrome that is not observed in other structurally characterized members. Active site variation is also observed, especially in comparison to its closest homologue cyt c'ß from the methane-oxidizing Methylococcus capsulatus Bath, which also lacks the cross-link. The phenylalanine "cap" which is presumed to control small ligand access to the distal heme iron is replaced with an arginine, reminiscent of the strictly conserved distal arginine in peroxidases and to the NH2OH-oxidizing cytochromes P460. A critical proton-transferring glutamate residue required for NH2OH oxidation is nevertheless missing in the active site. This in part explains the inability of cyt c'ß-Met to oxidize NH2OH. Our structure also rationalizes the absence of a methionyl cross-link, although the side chain's spatial position in the structure does not eliminate the possibility that it could form under certain conditions.

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Crystal structure of a hypothetical protein from Giardia lamblia. Corrigendum.

The name of one of the authors in Beard et al. [(2022), Acta Cryst. F78, 59-65] is corrected.

Crystal structure of an inorganic pyrophosphatase from Chlamydia trachomatis D/UW-3/Cx.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections globally and is one of the most commonly reported infections in the United States. There is a need to develop new therapeutics due to drug resistance and the failure of current treatments to clear persistent infections. Structures of potential C. trachomatis rational drug-discovery targets, including C. trachomatis inorganic pyrophosphatase (CtPPase), have been determined by the Seattle Structural Genomics Center for Infectious Disease. Inorganic pyrophosphatase hydrolyzes inorganic pyrophosphate during metabolism. Furthermore, bacterial inorganic pyrophosphatases have shown promise for therapeutic discovery. Here, a 2.2 Šresolution X-ray structure of CtPPase is reported. The crystal structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics for C. trachomatis.

Crystal structure of betaine aldehyde dehydrogenase from Burkholderia pseudomallei.

Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei.

Crystal structure of a hypothetical protein from Giardia lamblia.

Giardiasis is the most prevalent diarrheal disease globally and affects humans and animals. It is a significant problem in developing countries, the number one cause of travelers' diarrhea and affects children and immunocompromised individuals, especially HIV-infected individuals. Giardiasis is treated with antibiotics (tinidazole and metronidazole) that are also used for other infections such as trichomoniasis. The ongoing search for new therapeutics for giardiasis includes characterizing the structure and function of proteins from the causative protozoan Giardia lamblia. These proteins include hypothetical proteins that share 30% sequence identity or less with proteins of known structure. Here, the atomic resolution structure of a 15.6 kDa protein was determined by molecular replacement. The structure has the two-layer αß-sandwich topology observed in the prototypical endoribonucleases L-PSPs (liver perchloric acid-soluble proteins) with conserved allosteric active sites containing small molecules from the crystallization solution. This article is an educational collaboration between Hampton University and the Seattle Structural Genomics Center for Infectious Disease.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.