Targeting Tuberculosis: Novel Scaffolds for Inhibiting Cytochrome bd Oxidase.

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.

Introduction of the Capsules environment to support further growth of the SBGrid structural biology software collection.

The expansive scientific software ecosystem, characterized by millions of titles across various platforms and formats, poses significant challenges in maintaining reproducibility and provenance in scientific research. The diversity of independently developed applications, evolving versions and heterogeneous components highlights the need for rigorous methodologies to navigate these complexities. In response to these challenges, the SBGrid team builds, installs and configures over 530 specialized software applications for use in the on-premises and cloud-based computing environments of SBGrid Consortium members. To address the intricacies of supporting this diverse application collection, the team has developed the Capsule Software Execution Environment, generally referred to as Capsules. Capsules rely on a collection of programmatically generated bash scripts that work together to isolate the runtime environment of one application from all other applications, thereby providing a transparent cross-platform solution without requiring specialized tools or elevated account privileges for researchers. Capsules facilitate modular, secure software distribution while maintaining a centralized, conflict-free environment. The SBGrid platform, which combines Capsules with the SBGrid collection of structural biology applications, aligns with FAIR goals by enhancing the findability, accessibility, interoperability and reusability of scientific software, ensuring seamless functionality across diverse computing environments. Its adaptability enables application beyond structural biology into other scientific fields.

Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Finding priority bacterial ribosomes for future structural and antimicrobial research based upon global RNA and protein sequence analysis.

Ribosome-targeting antibiotics comprise over half of antibiotics used in medicine, but our fundamental knowledge of their binding sites is derived primarily from ribosome structures of non-pathogenic species. These include Thermus thermophilus, Deinococcus radiodurans and the archaean Haloarcula marismortui, as well as the commensal and sometimes pathogenic organism, Escherichia coli. Advancements in electron cryomicroscopy have allowed for the determination of more ribosome structures from pathogenic bacteria, with each study highlighting species-specific differences that had not been observed in the non-pathogenic structures. These observed differences suggest that more novel ribosome structures, particularly from pathogens, are required for a more accurate understanding of the level of diversity of the entire bacterial ribosome, with the potential of leading to innovative advancements in antibiotic research. In this study, high accuracy covariance and hidden Markov models were used to annotate ribosomal RNA and protein sequences respectively from genomic sequence, allowing us to determine the underlying ribosomal sequence diversity using phylogenetic methods. This analysis provided evidence that the current non-pathogenic ribosome structures are not sufficient representatives of some pathogenic bacteria, such as Campylobacter pylori, or of whole phyla such as Bacteroidota (Bacteroidetes).

Methods to Assess Chemokine Binding and Anti-chemotactic Activity of Virus Proteins.

Chemokines are key instigators of inflammatory and immune responses. Viruses can suppress these responses by secreting proteins that interfere with chemokine action. These proteins bind to chemokines and block the host's ability to recruit immune cells to sites of infection, thus facilitating virus replication and spread. When produced recombinantly, chemokine binding proteins provide a formidable resource to deploy against human disease. Here, we describe an enzyme-linked immunosorbent inhibition assay and a chemotaxis inhibition assay that are employed to assess the chemokine binding strength and anti-chemotactic activity of viral proteins. These assays are quick and reproducible, and are thus ideal for screening putative or modified chemokine binding proteins as the first step in their development as therapeutics.

Synthesis and Biological Evaluation of Aurachin D Analogues as Inhibitors of Mycobacterium tuberculosis Cytochrome bd Oxidase.

A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 µM).

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.

An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

Optimisation of Neuraminidase Expression for Use in Drug Discovery by Using HEK293-6E Cells.

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

The Immune Response to SARS-CoV-2 and Variants of Concern.

At the end of 2019 a newly emerged betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of an outbreak of severe pneumonia, subsequently termed COVID-19, in a number of patients in Wuhan, China. Subsequently, SARS-CoV-2 rapidly spread globally, resulting in a pandemic that has to date infected over 200 million individuals and resulted in more than 4.3 million deaths. While SARS-CoV-2 results in severe disease in 13.8%, with increasing frequency of severe disease with age, over 80% of infections are asymptomatic or mild. The immune response is an important determinant of outcome following SARS-CoV-2 infection. While B cell and T cell responses are associated with control of infection and protection against subsequent challenge with SARS-CoV-2, failure to control viral replication and the resulting hyperinflammation are associated with severe COVID-19. Towards the end of 2020, several variants of concern emerged that demonstrate increased transmissibility and/or evasion of immune responses from prior SARS-CoV-2 infection. This article reviews what is known about the humoral and cellular immune responses to SARS-CoV-2 and how mutation and structural/functional changes in the emerging variants of concern impact upon the immune protection from prior infection or vaccination.

The cryo-EM structure of the bd oxidase from M. tuberculosis reveals a unique structural framework and enables rational drug design to combat TB.

New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis.

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.

The post-lockdown period should be used to acquire effective therapies for future resurgence in SARS-Cov-2 infections.

COVID-19 will be with us through the remainder of 2020 and almost certainly beyond. New Zealand needs a viable strategy to protect its populace until a vaccine is developed and in wide use. Until that time, it makes sense to protect the population by putting in place treatments that will be safe and effective, such as the use of convalescent sera and the use of direct-acting anti-virals. These treatments should be sourced externally or made locally, but steps in this direction must now begin as the lockdown ends. New Zealand has the scientists, the facilities and the will to make this happen, but the support of the government and the population will be needed if this plan is to succeed.

Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3GCTD and in that of full-length wild-type APOBEC3G.

Reconstitution of CRISPR adaptation in vitro and its detection by PCR.

CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR-Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2-3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR-Cas systems, including the roles of accessory proteins.

Detection of D-glutamate production from the dual Function enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis.

D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen et al., 2016 ). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.

New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a firefly-like luciferase but an entirely new luciferin.

The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the firefly. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purified luciferase enzyme is in the same protein family as firefly luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely different to that of the firefly and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confirmed by chemical synthesis and bioluminescence assays. These findings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally different luciferins.

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.