The Importance of Epigallocatechin as a Scaffold for Drug Development against Flaviviruses.

Arboviruses such as Dengue, yellow fever, West Nile, and Zika are flaviviruses vector-borne RNA viruses transmitted biologically among vertebrate hosts by blood-taking vectors. Many flaviviruses are associated with neurological, viscerotropic, and hemorrhagic diseases, posing significant health and socioeconomic concerns as they adapt to new environments. Licensed drugs against them are currently unavailable, so searching for effective antiviral molecules is still necessary. Epigallocatechin molecules, a green tea polyphenol, have shown great virucidal potential against flaviviruses, including DENV, WNV, and ZIKV. The interaction of EGCG with the viral envelope protein and viral protease, mainly identified by computational studies, describes the interaction of these molecules with viral proteins; however, how the viral NS2B/NS3 protease interacts with epigallocatechin molecules is not yet fully deciphered. Consequently, we tested the antiviral potential of two epigallocatechin molecules (EGC and EGCG) and their derivative (AcEGCG) against DENV, YFV, WNV, and ZIKV NS2B/NS3 protease. Thus, we assayed the effect of the molecules and found that a mixture of the molecules EGC (competitive) and EGCG (noncompetitive) inhibited the virus protease of YFV, WNV, and ZIKV more effectively with IC50 values of 1.17 ± 0.2 µM, 0.58 ± 0.07 µM, and 0.57 ± 0.05 µM, respectively. As these molecules fundamentally differ in their inhibitory mode and chemical structure, our finding may open a new line for developing more effective allosteric/active site inhibitors to combat flaviviruses infection.

Discovery of All-d-Peptide Inhibitors of SARS-CoV-2 3C-like Protease.

During the replication process of SARS-CoV-2, the main protease of the virus [3-chymotrypsin-like protease (3CLpro)] plays a pivotal role and is essential for the life cycle of the pathogen. Numerous studies have been conducted so far, which have confirmed 3CLpro as an attractive drug target to combat COVID-19. We describe a novel and efficient next-generation sequencing (NGS) supported phage display selection strategy for the identification of a set of SARS-CoV-2 3CLpro targeting peptide ligands that inhibit the 3CL protease, in a competitive or noncompetitive mode, in the low µM range. From the most efficient l-peptides obtained from the phage display, we designed all-d-peptides based on the retro-inverso (ri) principle. They had IC50 values also in the low µM range and in combination, even in the sub-micromolar range. Additionally, the combination with Rutinprivir decreases 10-fold the IC50 value of the competitive inhibitor. The inhibition modes of these d-ri peptides were the same as their respective l-peptide versions. Our results demonstrate that retro-inverso obtained all-d-peptides interact with high affinity and inhibit the SARS-CoV-2 3CL protease, thus reinforcing their potential for further development toward therapeutic agents. The here described d-ri peptides address limitations associated with current l-peptide inhibitors and are promising lead compounds. Further optimization regarding pharmacokinetic properties will allow the development of even more potent d-peptides to be used for the prevention and treatment of COVID-19.

Development of an α-synuclein fibril and oligomer specific tracer for diagnosis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.

The development of specific disease-associated PET tracers is one of the major challenges, the realization of which in neurodegenerative diseases would enable not only the efficiency of diagnosis but also support the development of disease-modifying therapeutics. Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by neuronal fibrillary inclusions composed of aggregated α-synuclein (α-syn). However, these deposits are not only found in PD, but also in other related diseases such as multiple system atrophy (MSA) and dementia with Lewy bodies (DLB), which are grouped under the term synucleinopathies. In this study, we used NGS-guided phage display selection to identify short peptides that bind aggregated α-syn. By surface plasmon resonance (SPR)-based affinity screening, we identified the peptide SVLfib-5 that recognizes aggregated α-syn with high complex stability and sequence specificity. Further analysis SPR showed that SVLfib-5 is not only specific for aggregated α-syn, but in particular recognizes fibrillary and oligomeric structures. Moreover, fluorescence microscopy of human brain tissue sections from PD, MSA, and DLB patients with SVLfib-5 allowed specific recognition of α-syn and a clear discrimination between diseased and non-diseased samples. These findings provide the basis for the further development of an α-syn PET tracer for early diagnosis and monitoring of disease progression and therapy progress.

A So-Far Overlooked Secondary Conformation State in the Binding Mode of SARS-CoV-2 Spike Protein to Human ACE2 and Its Conversion Rate Are Crucial for Estimating Infectivity Efficacy of the Underlying Virus Variant.

Since its outbreak in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been multiple times replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates. In order to find an explanation for why SARS-CoV-2 and its emerging mutated versions showed enhanced transmission efficiency compared with SARS-CoV (2002), an enhanced binding affinity of the spike protein to human angiotensin converting enzyme 2 (hACE2) has been proposed by crystal structure analysis and was identified in cell culture models. Kinetic analysis of the interaction of various spike protein constructs with hACE2 was considered to be best described by a Langmuir-based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with hACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of ~190 (primary versus secondary state) with an overall equilibrium dissociation constant (KD) of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wild type ("wt"; Wuhan strain) but also found in the B.1.1.7 variant, where its transition rate is 5-fold increased. IMPORTANCE The current SARS-CoV-2 pandemic is characterized by the high infectivity of SARS-CoV-2 and its derived variants of concern (VOCs). It has been widely assumed that the reason for its increased cell entry compared with SARS-CoV (2002) is due to alterations in the viral spike protein, where single amino acid residue substitutions can increase affinity for hACE2. So far, the interaction of a single unit of the CoV-2 spike protein has been described using the 1:1 Langmuir interaction kinetic. However, we demonstrate here that there is a secondary state binding step that may be essential for novel VOCs in order to further increase their infectivity. These findings are important for quantitatively understanding the infection process of SARS-CoV-2 and characterization of emerging SARS-CoV-2 variants of spike proteins. Thus, they provide a tool for predicting the potential infectivity of the respective viral variants based on secondary rate transition and secondary complex stability.

Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo.

Parkinson's disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.

Phage Display-Derived Compounds Displace hACE2 from Its Complex with SARS-CoV-2 Spike Protein.

Severe respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious beta-class coronavirus. Although vaccinations have shown high efficacy, the emergence of novel variants of concern (VOCs) has already exhibited traits of immune evasion. Thus, the development of tailored antiviral medications for patients with incomplete, inefficient, or non-existent immunization, is essential. The attachment of viral surface proteins to the cell surface is the first crucial step in the viral replication cycle, which for SARS-CoV-2 is mediated by the high affinity interaction of the viral trimeric spike with the host cell surface-located human angiotensin converting enzyme-2 (hACE2). Here, we used a novel and efficient next generation sequencing (NGS) supported phage display strategy for the selection of a set of SARS-CoV-2 receptor binding domain (RBD)-targeting peptide ligands that bind to the target protein with low µM to nM dissociation constants. Compound CVRBDL-3 inhibits the SARS-CoV-2 spike protein association to hACE2 in a concentration-dependent manner for pre- as well as post-complex formation conditions. Further rational optimization yielded a CVRBDL-3 based divalent compound, which demonstrated inhibitory efficacy with an IC50 value of 47 nM. The obtained compounds were not only efficient for the different spike constructs from the originally isolated "wt" SARS-CoV-2, but also for B.1.1.7 mutant trimeric spike protein. Our work demonstrates that phage display-derived peptide ligands are potential fusion inhibitors of viral cell entry. Moreover, we show that rational optimization of a combination of peptide sequences is a potential strategy in the further development of therapeutics for the treatment of acute COVID-19.

Ligand-Induced Stabilization of the Native Human Superoxide Dismutase 1.

A common characteristic of familial (fALS) and sporadic amyotrophic lateral sclerosis (sALS) is the accumulation of aberrant proteinaceous species in the motor neurons and spinal cord of ALS patients-including aggregates of the human superoxide dismutase 1 (hSOD1). hSOD1 is an enzyme that occurs as a stable dimeric protein with several post-translational modifications such as the formation of an intramolecular disulfide bond and the acquisition of metal cofactors that are essential for enzyme activity and further contribute to protein stability. Some mutations and/or destabilizing factors promote hSOD1 misfolding, causing neuronal death. Aggregates containing misfolded wild-type hSOD1 have been found in the spinal cords of sALS as well as in non-hSOD1 fALS patients, leading to the hypothesis that hSOD1 misfolding is a common part of the ALS pathomechanism. Therefore, stabilizing the native conformation of SOD1 may be a promising approach to prevent the formation of toxic hSOD1 species and thus ALS pathogenesis. Here, we present the 16-mer peptide S1VL-21 that interferes with hSOD1 aggregation. S1VL-21 was identified by phage display selection with the native conformation of hSOD1 as a target. Several methods such as microscale thermophoresis (MST) measurements, aggregation assays, and cell viability assays revealed that S1VL-21 has a micromolar binding affinity to native hSOD1 and considerably reduces the formation of hSOD1 aggregates. This present work therefore provides the first important data on a potential lead compound for hSOD1-related drug development for ALS therapy.