Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate and harbors a Zn2+ ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives. High-resolution crystal structures of GmhA complexed to two N-formyl hydroxamate inhibitors confirmed the binding interactions to a central Zn2+ ion coordination site. Some of these compounds were found to be nanomolar inhibitors of GmhA. While devoid of HepG2 cytotoxicity and antibacterial activity of their own, they demonstrated in vitro lipopolysaccharide heptosylation inhibition in Enterobacteriaceae as well as the potentiation of erythromycin and rifampicin in a wild-type Escherichia coli strain. These inhibitors pave the way for a novel treatment of Gram-negative infections.

A novel fluorescent reporter sensitive to serine mis-incorporation.

High-fidelity translation was considered a requirement for living cells. The frozen accident theory suggested that any deviation from the standard genetic code should result in the production of so much mis-made and non-functional proteins that cells cannot remain viable. Studies in bacterial, yeast, and mammalian cells show that significant levels of mistranslation (1-10% per codon) can be tolerated or even beneficial under conditions of oxidative stress. Single tRNA mutants, which occur naturally in the human population, can lead to amino acid mis-incorporation at a codon or set of codons. The rate or level of mistranslation can be difficult or impossible to measure in live cells. We developed a novel red fluorescent protein reporter that is sensitive to serine (Ser) mis-incorporation at proline (Pro) codons. The mCherry Ser151Pro mutant is efficiently produced in Escherichia coli but non-fluorescent. We demonstrated in cells and with purified mCherry protein that the fluorescence of mCherry Ser151Pro is rescued by two different tRNASer gene variants that were mutated to contain the Pro (UGG) anticodon. Ser mis-incorporation was confirmed by mass spectrometry. Remarkably, E. coli tolerated mistranslation rates of ~10% per codon with negligible reduction in growth rate. Conformational sampling simulations revealed that the Ser151Pro mutant leads to significant changes in the conformational freedom of the chromophore precursor, which is indicative of a defect in chromophore maturation. Together our data suggest that the mCherry Ser151 mutants may be used to report Ser mis-incorporation at multiple other codons, further expanding the ability to measure mistranslation in living cells.

An Inhibitor-in-Pieces Approach to DAHP Synthase Inhibition: Potent Enzyme and Bacterial Growth Inhibition.

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate biosynthetic pathway and is an antimicrobial target. We used an inhibitor-in-pieces approach, based on the previously reported inhibitor DAHP oxime, to screen inhibitor fragments in the presence and absence of glycerol 3-phosphate to occupy the distal end of the active site. This led to DAHP hydrazone, the most potent inhibitor to date, Ki = 10 ± 1 nM. Three trifluoropyruvate (TFP)-based inhibitor fragments were efficient inhibitors with ligand efficiencies of up to 0.7 kcal mol-1/atom compared with 0.2 kcal mol-1/atom for a typical good inhibitor. The crystal structures showed the TFP-based inhibitors binding upside down in the active site relative to DAHP oxime, providing new avenues for inhibitor development. The ethyl esters of TFP oxime and TFP semicarbazone prevented E. coli growth in culture with IC50 = 0.21 ± 0.01 and 0.77 ± 0.08 mg mL-1, respectively. Overexpressing DAHP synthase relieved growth inhibition, demonstrating that DAHP synthase was the target. Growth inhibition occurred in media containing aromatic amino acids, suggesting that growth inhibition was due to depletion of some other product(s) of the shikimate pathway, possibly folate.

Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2.

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

NeuNAc Oxime: A Slow-Binding and Effectively Irreversible Inhibitor of the Sialic Acid Synthase NeuB.

NeuB is a bacterial sialic acid synthase used by neuroinvasive bacteria to synthesize N-acetylneuraminate (NeuNAc), helping them to evade the host immune system. NeuNAc oxime is a potent slow-binding NeuB inhibitor. It dissociated too slowly to be detected experimentally, with initial estimates of its residence time in the active site being >47 days. This is longer than the lifetime of a typical bacterial cell, meaning that inhibition is effectively irreversible. Inhibition data fitted well to a model that included a pre-equilibration step with a Ki of 36 µM, followed by effectively irreversible conversion to an E*·I complex, with a k2 of 5.6 × 10-5 s-1. Thus, the inhibitor can subvert ligand release and achieve extraordinary residence times in spite of a relatively modest initial dissociation constant. The crystal structure showed the oxime functional group occupying the phosphate-binding site normally occupied by the substrate PEP and the tetrahedral intermediate. There was an ≈10% residual rate at high inhibitor concentrations regardless of how long NeuB and NeuNAc oxime were preincubated together. However, complete inhibition was achieved by incubating NeuNAc oxime with the actively catalyzing enzyme. This requirement for the enzyme to be actively turning over for the inhibitor to bind to the second subunit demonstrated an important role for intersubunit communication in the inhibitory mechanism.