Amantadine Inhibits SARS-CoV-2 In Vitro.

Since the SARS-CoV-2 pandemic started in late 2019, the search for protective vaccines and for drug treatments has become mandatory to fight the global health emergency. Travel restrictions, social distancing, and face masks are suitable counter measures, but may not bring the pandemic under control because people will inadvertently or at a certain degree of restriction severity or duration become incompliant with the regulations. Even if vaccines are approved, the need for antiviral agents against SARS-CoV-2 will persist. However, unequivocal evidence for efficacy against SARS-CoV-2 has not been demonstrated for any of the repurposed antiviral drugs so far. Amantadine was approved as an antiviral drug against influenza A, and antiviral activity against SARS-CoV-2 has been reasoned by analogy but without data. We tested the efficacy of amantadine in vitro in Vero E6 cells infected with SARS-CoV-2. Indeed, amantadine inhibited SARS-CoV-2 replication in two separate experiments with IC50 concentrations between 83 and 119 µM. Although these IC50 concentrations are above therapeutic amantadine levels after systemic administration, topical administration by inhalation or intranasal instillation may result in sufficient amantadine concentration in the airway epithelium without high systemic exposure. However, further studies in other models are needed to prove this hypothesis.

Studies on the dissociation of botulinum neurotoxin type A complexes.

UNLABELLED: The neurotoxins produced by the various strains of the anaerobic bacterium Clostridium botulinum naturally occur associated with complexing proteins which serve to protect the neurotoxins from the harsh environment of the mammalian gastrointestinal tract during bacterial invasion of the host. Three different complex species with the discrete sizes 19S (900 kDa, LL complex), 16S (500 kDa, L complex) and 12S (300 kDa, M complex) may be isolated from C. botulinum type A cultures. However, to affect their target cells these complexes must dissociate releasing the free 150 kDa neurotoxin. This study assesses the stability of these Clostridium botulinum neurotoxin serotype A (BoNT/A) complexes and identifies factors which influence their dissociation. The knowledge gained with purified toxin complexes was subsequently employed to analyze the presence of such complexes in the freeze or spray-dried commercial BoNT/A products Botox and Dysport in comparison to the complexing protein free product Xeomin. Purified 900 kDa and 500 kDa toxin complex preparations show a pH and time dependent release of the 150 kDa neurotoxin with a half-life of less than a minute at pH values >7.0. At pH values of 6.25 or less, the complexes are stable. Furthermore, dilution of concentrated 900 kDa complexes leads to dissociation into 500 kDa, neurotoxin containing complexes. Addition of sodium chloride as contained in isotonic saline leads to further disruption of these complexes resulting in the release of the free 150 kDa neurotoxin. Examination of the commercial botulinum neurotoxin products Botox and Dysport using the same analytical procedures leads to the same conclusion: the dilution, drying and reconstitution processes of these products lead to a complete dissociation of 900 kDa complexes and 85% or more of neurotoxin are present in free form. CONCLUSION: BoNT A toxin complexes have evolved to quickly respond to specific environmental changes by efficient release of the neurotoxin. During pharmaceutical production and reconstitution of BoNT A products, the same principles effect the quantitative dissociation of 900 kDa complexes and release of free neurotoxin prior to injection into target tissues.

Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells.

It was shown previously [Riedinger, H. J., van Betteraey-Nikoleit, M & Probst, H. (2002) Eur. J. Biochem.269, 2383-2393] that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.