Kurt Krause

The cysteine 3C-like proteases (3CLpro) of caliciviruses, coronaviruses, and picornaviruses are essential for viral replication. In this study, we report the development of potent broad-spectrum peptidomimetic antiviral agents that target the 3CLpro of caliciviruses (NS6), coronaviruses (Mpro), and a picornavirus (3C). Based upon previously reported inhibitors, a small library of compounds was designed, synthesized and tested to identify a core structure, which was then derivatized with a focus upon P3 and P4 positions to afford new inhibitors with improved potency against the respective viral enzymes and enhanced binding as determined by X-ray crystallography. These compounds were tested against a range of viruses in culture, revealing minimal toxicity while exhibiting broad-spectrum potent nanomolar activities against noroviruses and several coronavirus species, including alpha and omicron variants of SARS-CoV-2 and Middle East Respiratory Syndrome virus (MERS).

Ambitious milestones set by the World Health Organisation (WHO) to end the tuberculosis epidemic by 2030 currently appear out of reach, and there remains an urgent need to develop more effective novel therapies. While exploring dipicolinic acid derivatives as putative glutamate racemase inhibitors, we recently discovered 6-nitropicolinamides as promising antituberculosis agents. SAR studies on the non-cytotoxic N-[4-(trifluoromethoxy)benzyl] hit 20 (MIC90 1.4 μM) confirmed the importance of the 6-nitro group and amide NH; side chain extension enhanced potency but reduced aqueous solubility, and some analogues were rapidly metabolised. The best new candidate (77: MIC90 0.30 μM) was well tolerated in mice and provided an adequate pharmacokinetic profile, although a time-kill assay indicated largely bacteriostatic activity. An analysis of its effects on cell wall lipids and mycolic acids revealed changes consistent with inhibiting arabinogalactan biosynthesis, and further testing against mutant or overexpressing mycobacterial strains identified the enzyme target as decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1).

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.

Importance: Despite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis . This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.

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.

The integration of Capsules within the SBGrid software-management platform marks a pivotal advancement in addressing the challenges of scientific software distribution, dependency management and computational reproducibility. 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.

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 angstrom and at 1.83 angstrom with a covalently bound high-affinity (IC50 = 0.37 mu M) 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.

Aotearoa New Zealand recently recorded its first case of Candida auris: a pathogen that is difficult to contain in healthcare settings. Since its first detection in 2009, cases of C. auris have become more widespread globally. In this Briefing we evaluate the risk of C. auris to New Zealanders and outline how best to control its spread. Transmission of C. auris occurs mainly through contact with contaminated surfaces and medical equipment. Standard hospital-grade disinfectants are not effective at killing C. auris: instead, agents with sporicidal activity are needed. We recommend that rigorous screening and surveillance measures are reinforced to prevent C. auris spread in New Zealand. Diagnostic microbiology laboratories should establish and validate methods for the screening of C. auris, including rapid identification of suspected isolates and pathways to ensure prompt susceptibility results are available. We recommend mandatory notification of confirmed C. auris cases to support control and epidemiological surveillance.

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 , and the archaean , as well as the commensal and sometimes pathogenic organism, . 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 , or of whole phyla such as Bacteroidota (Bacteroidetes).