A Nonprofit Drug Development Model Is Part of the Antimicrobial Resistance (AMR) Solution.

Antibiotics underpin modern medicine and are critical for pandemic preparedness. Push funding has revitalized the preclinical antimicrobial resistance (AMR) pipeline and government funding via CARB-X and BARDA, as well as private sector-led investment via the AMR Action Fund, will help several new antibiotics obtain regulatory approval. Nevertheless, revenues generated by new antibiotics are not considered sufficiently profitable by commercial developers to address unmet need. The question remains: Who could viably fund development and secure global equitable access for new antibiotics? Public health need should be the primary driver for antibiotic development. Improved prioritization and government oversight by funders who allocate public resources are a needed first step. In this framework, nonprofit research and development organizations, with support from public funders, and unconstrained by commercial profitability requirements are well positioned to work with public and private actors to viably provide new antibiotics to all in need.

Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

Selective export of transmembrane proteins from the endoplasmic reticulum (ER) relies on recognition of cytosolic-domain-localized transport signals by the Sec24 subunit of the COPII vesicle coat. Human cells express four Sec24 isoforms, termed Sec24A, Sec24B, Sec24C and Sec24D that are differentially required for selective, signal-mediated ER export of transmembrane proteins. By contrast, luminally exposed glycosylphosphatidylinositol (GPI)-anchored membrane proteins cannot bind directly to Sec24 and must either use membrane-spanning cargo receptors or alternative mechanisms for ER export. Little is known about the mechanism underlying export of GPI-anchored proteins from the ER in higher eukaryotes. Using siRNA-based silencing, we identified that ER-to-Golgi transport of the human GPI-anchored protein CD59 requires Sec24, with preference for the Sec24C and Sec24D isoforms, and the recycling transmembrane protein complex p24-p23 that exhibited the same Sec24C-Sec24D isoform preference for ER export. Co-immunoprecipitation indicated unprecedented physical interaction of CD59 as well as a GFP-folate-receptor-GPI-anchor hybrid with a p24-p23 complex. Density gradient centrifugation revealed co-partitioning of CD59 and p24-p23 into biosynthetically early lipid raft fractions, and CD59 transport to the Golgi was cholesterol dependent. The results suggest that the 24p-23p complex acts as a cargo receptor for GPI-anchored proteins by facilitating their export from the ER in a Sec24-isoform-selective manner involving lipid rafts as early sorting platforms.

Analysis of the clinical antibacterial and antituberculosis pipeline.

This analysis of the global clinical antibacterial pipeline was done in support of the Global Action Plan on Antimicrobial Resistance. The study analysed to what extent antibacterial and antimycobacterial drugs for systemic human use as well as oral non-systemic antibacterial drugs for Clostridium difficile infections were active against pathogens included in the WHO priority pathogen list and their innovativeness measured by their absence of cross-resistance (new class, target, mode of action). As of July 1, 2018, 30 new chemical entity (NCE) antibacterial drugs, ten biologics, ten NCEs against Mycobacterium tuberculosis, and four NCEs against C difficile were identified. Of the 30 NCEs, 11 are expected to have some activity against at least one critical priority pathogen expressing carbapenem resistance. The clinical pipeline is dominated by derivatives of established classes and most development candidates display limited innovation. New antibacterial drugs without pre-existing cross-resistance are under-represented and are urgently needed, especially for geographical regions with high resistance rates among Gram-negative bacteria and M tuberculosis.

Regulation of a COPII component by cytosolic O-glycosylation during mitosis.

Endoplasmic reticulum (ER)-to-Golgi transport is blocked in mammalian cells during mitosis; however, the mechanism underlying this blockade remains unknown. Since COPII proteins are involved in this transport pathway, we investigated at the biochemical level post-translational modifications of COPII components during the course of mitosis that could be linked to inhibition of ER-to-Golgi transport. By comparing biochemical properties of cytosolic COPII components during interphase and mitosis, we found that Sec24p isoforms underwent post-translational modifications resulting in an increase in their apparent molecular weight. No such modification was observed for the other COPII components Sec23p, Sec13p, Sec31p or Sar1p. Analyzing in more details Sec24p isoforms in interphase and mitotic conditions, we found that the interphase form of Sec24p was O-N-acetylglucosamine modified, a feature lost upon entering into mitosis. This mitotic deglycosylation was coupled to Sec24p phosphorylation, a feature likely responsible for the increase in apparent molecular weight of these molecules. These modifications correlated with an alteration in the membrane binding properties of Sec24p. These data suggest that when entering into mitosis, the COPII component Sec24p is simultaneously deglycosylated and phosphorylated, a process which may contribute to the observed mitotic ER-to-Golgi traffic block.

Role of Sec24 isoforms in selective export of membrane proteins from the endoplasmic reticulum.

Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast. Human cells express four Sec24 isoforms, but their role is unknown. Here, we report the differential effects of Sec24 isoform-specific silencing on the transport of the membrane reporter protein ERGIC-53 (ER-Golgi intermediate compartment-53) carrying the cytosolic ER export signals di-phenylalanine, di-tyrosine, di-leucine, di-isoleucine, di-valine or terminal valine. Knockdown of single Sec24 isoforms showed dependence of di-leucine-mediated transport on Sec24A, but transport mediated by the other signals was not affected. By contrast, double knockdown of Sec24A with one of the other three Sec24 isoforms impaired all aromatic/hydrophobic signal-dependent transport. Double knockdown of Sec24B/C or Sec24B/D preferentially affected di-leucine-mediated transport, whereas knockdown of Sec24C/D affected di-isoleucine- and valine-mediated transport. The isoform-selective transport correlated with binding preferences of the signals for the corresponding isoforms in vitro. Thus, human Sec24 isoforms expand the repertoire of cargo for signal-mediated ER export, but are in part functionally redundant.

Inhibitors of bacterial virulence identified in a surrogate host model.

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using non-animal host models.

Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export.

Export of membrane proteins from the ER is believed to be selective and require transport signals, but the identity of such signals has remained elusive. The recycling type I membrane protein ERGIC-53 carries a C-terminal diphenylalanine motif that is required for efficient ER export. Here we show that this motif can be functionally substituted by a single phenylalanine or tyrosine at position -2, two leucines or isoleucines at position -1 and -2 or a single valine at position -1. These motifs are common among mammalian type I membrane proteins. A single C-terminal valine, but none of the other motifs, accelerates transport of inefficiently exported reporter constructs and hence operates as an export signal. The valine signal is position, but not context, dependent. All transport motifs mediate COPII binding in vitro with distinct preferences for the COPII subunits Sec23p, Sec24Bp, Sec24Cp and p125. These results suggest that cytoplasmic C-terminal amino-acid motifs, either alone or in conjunction with other transport determinants, accelerate ER export of numerous type I and probably polytopic membrane proteins by mediating interaction with COPII coat components.