Bacterial infectious diseases remain a major global health threat due to increasing antibiotic resistance. Further complicating effective treatment, some pathogens such as Staphylococcus aureus persist within host cells. Peptidoglycan hydrolases (PGHs), including bacteriophage-derived endolysins, represent a promising class of novel antimicrobials due to their rapid bacteriolytic activity and low risk of resistance emergence. However, their clinical application is limited by an unfavourable PK profile and inefficient delivery into infected host cells. Here, we engineered extracellular vesicles (EVs) for the targeted, intracellular delivery of GH15, a novel endolysin against S. aureus. GH15-loaded EVs, functionalized with antibodies targeting αvβ3 integrin on bacterially infected cells, efficiently delivered GH15 into S. aureus-positive endothelial cells and macrophages in vitro and eliminated intracellular bacteria. In addition, GH15-loaded EVs promoted the clearance of intracellular bacteria in macrophages of infected zebrafish larvae in vivo. Our findings establish EVs as effective vehicles for intracellular delivery of antimicrobial enzymes and highlight their potential for the treatment of intracellular bacterial infections.

Despite vaccine availability, the morbilliviruses measles virus and canine distemper virus (CDV) are still causing major health impairments in human and animal populations. Here, we identified two potent, neutralizing single domain antibodies directed against the tetrameric receptor binding (H) protein of CDV. Structural analyses spotlighted two vulnerable sites within the H protein. While the first overlaps with the receptor binding site, the second encompasses amino acid residues of two protomers located at the distal dimeric head interface, which supports distinct mechanisms of neutralization. Upon application of an engineered tetravalent and biparatopic antibody, ferrets were protected at a remarkably low antibody dose (1 mg/kg) administered intra-peritoneally on days 3 and 7 post-exposure of a lethal CDV challenge. Collectively, this study spotlights the power of integrating multiple mechanisms of neutralization in a single format and provides a roadmap to design next-generation therapeutics against morbilliviral infections as well as other infectious pathogens. Measles virus and canine distemper virus (CDV) are still causing health impairments in humans and animals. Here, the authors develop a synthetic antibody protecting ferrets from lethal CDV infections, providing a roadmap for antiviral drug design.

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico , this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro . To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators. ### Competing Interest Statement The authors have declared no competing interest.

Lefleuganan, a clinical stage drug candidate for the treatment of cutaneous leishmaniasis, is a synthetic nonapeptide inspired by the natural antimicrobial peptide leucinostatin A, exhibiting excellent antiprotozoal activity in the low nM range. Lefleuganan shows strongly reduced acute toxicity, making it amenable for clinical use. Here, using a broad set of in vivo and in vitro measurements using purified enzymes, we find that leucinostatin A, but not lefleuganan, specifically targets the mitochondrial ATP synthase, inhibiting ATP synthesis by the human, bovine, and yeast enzyme in the nanomolar range. In a structure-activity relationship study covering the chemical space between the two compounds, hydroxyleucine at position 7 in leucinostatin A is identified as the key responsible moiety for specific ATP synthase inhibition and systemic toxicity. Our data suggest that efficient antiprotozoal activity of these class of compounds is mediated by efficient energetic uncoupling of negatively charged membranes.

Targeted protein degradation (TPD) has revolutionized drug discovery by selectively eliminating specific proteins within and outside the cellular context. Over the past two decades, TPD has expanded its focus beyond well-established targets, exploring diverse proteins beyond cancer-related ones. This evolution extends the potential of TPD to various diseases. Notably, TPD can target proteins at demanding locations, such as the extracellular matrix (ECM) and cellular membranes, presenting both opportunities and challenges for future research. In this review, we comprehensively examine the exciting opportunities in the burgeoning field of TPD, highlighting different targets, their cellular environment, and innovative strategies for modern drug discovery.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like cysteine protease (PLpro) represents one of only two essential cysteine proteases involved in the regulation of viral replication. It, therefore, qualifies as a promising therapeutic target for the development of antiviral agents. We identified a previously synthesized protease inhibitor, resulting from an earlier project, as a PLpro inhibitor and crafted a structure-activity relationship around the hit, leading to the more potent inhibitors ZHAWOC6941 (17h) and ZHAWOC25153 (17o) displaying IC50 values of 8 and 7 µM, respectively. The two compounds represent a new class of PLpro inhibitors and, with single-digit micromolar IC50 values, are comparable to inhibitors found in the literature.

Canine distemper virus (CDV) is an enveloped RNA morbillivirus that triggers respiratory, enteric, and high incidence of severe neurological disorders. CDV induces devastating outbreaks in wild and endangered animals as well as in domestic dogs in countries associated with suboptimal vaccination programs. The receptor-binding tetrameric attachment (H)-protein is part of the morbilliviral cell entry machinery. Here, we present the cryo-electron microscopy (cryo-EM) structure and supramolecular organization of the tetrameric CDV H-protein ectodomain. The structure reveals that the morbilliviral H-protein is composed of three main domains: stalk, neck, and heads. The most unexpected feature was the inherent asymmetric architecture of the CDV H-tetramer being shaped by the neck, which folds into an almost 90° bent conformation with respect to the stalk. Consequently, two non-contacting receptor-binding H-head dimers, which are also tilted toward each other, are located on one side of an intertwined four helical bundle stalk domain. Positioning of the four protomer polypeptide chains within the neck domain is guided by a glycine residue (G158), which forms a hinge point exclusively in two protomer polypeptide chains. Molecular dynamics simulations validated the stability of the asymmetric structure under near physiological conditions and molecular docking showed that two receptor-binding sites are fully accessible. Thus, this spatial organization of the CDV H-tetramer would allow for concomitant protein interactions with the stalk and head domains without steric clashes. In summary, the structure of the CDV H-protein ectodomain provides new insights into the morbilliviral cell entry system and offers a blueprint for next-generation structure-based antiviral drug discovery.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

SUMOylation is a reversible post–translational modification (PTM) involving covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. Dysregulation of SUMOylation and deSUMOylation results in cellular malfunction and is linked to various diseases, such as cancer. Sentrin-specific proteases (SENPs) were identified for the maturation of SUMOs and the deconjugation of SUMOs from their substrate proteins. Hence, this is a promising target tackling the dysregulation of the SUMOylation process. Herein, we report the discovery of a novel protein-protein interaction (PPI) inhibitor for SENP1-SUMO1 by virtual screening and subsequent medicinal chemistry optimization of the hit molecule. The optimized inhibitor ZHAWOC8697 showed IC50 values of 8.6 μM against SENP1 and 2.3 μM against SENP2. With a photo affinity probe the SENP target was validated. This novel SENP inhibitor represents a new valuable tool for the study of SUMOylation processes and the SENP-associated development of small molecule-based treatment options.

BACKGROUND:Listeria monocytogenes (Lm) is a bacterial pathogen of major concern for humans and ruminants due to its neuroinvasive potential and its ability to cause deadly encephalitis (neurolisteriosis). On one hand, polymorphonuclear neutrophils (PMN) are key players in the defense against Lm, but on the other hand intracerebral infiltration with PMN is associated with significant neural tissue damage. Lm-PMN interactions in neurolisteriosis are poorly investigated, and factors inducing PMN chemotaxis to infectious foci containing Lm in the central nervous system (CNS) remain unidentified.METHODS:In this study, we assessed bovine PMN chemotaxis towards Lm and supernatants of infected endogenous brain cell populations in ex vivo chemotaxis assays, to identify chemotactic stimuli for PMN chemotaxis towards Lm in the brain. In addition, microglial secretion of IL-8 was assessed both ex vivo and in situ.RESULTS:Our data show that neither Lm cell wall components nor intact bacteria elicit chemotaxis of bovine PMN ex vivo. Moreover, astrocytes and neural cells fail to induce bovine PMN chemotaxis upon infection. In contrast, supernatant from Lm infected microglia readily induced chemotaxis of bovine PMN. Microglial expression and secretion of IL-8 was identified during early Lm infection in vitro and in situ, although IL-8 blocking with a specific antibody could not abrogate PMN chemotaxis towards Lm infected microglial supernatant.CONCLUSIONS:These data provide evidence that host-derived rather than bacterial factors trigger PMN chemotaxis to bacterial foci in the CNS, that microglia have a primary role as initiators of bovine PMN chemotaxis into the brain during neurolisteriosis and that blockade of these factors could be a therapeutic target to limit intrathecal PMN chemotaxis and PMN associated damage in neurolisteriosis.

Peptides comprising many hydrophobic amino acids are almost insoluble under physiological buffer conditions, which complicates their structural analysis. To investigate the three-dimensional structure of the hydrophobic leucinostatin derivative ZHAWOC6027, the previously developed host lattice display technology was applied. Two designed ankyrin-repeat proteins (DARPins) recognizing a biotinylated ZHAWOC6027 derivative were selected from a diverse library by ribosome display under aqueous buffer conditions. ZHAWOC6027 was immobilized by means of the DARPin in the host lattice and the structure of the complex was determined by X-ray diffraction. ZHAWOC6027 adopts a distorted α-helical conformation. Comparison with the structures of related compounds that have been determined in organic solvents reveals elevated flexibility of the termini, which might be functionally important.

Leucinostatins are fascinating natural substances, with high potency against many pathogens, but their acute toxicity has prevented therapeutic use. Michael Adams, Rainer Riedl, and co-workers demonstrate in the Research Article on page 15613 how these can be converted into synthetically more accessible and more selective antiprotozoal agents with broad efficacy and undetectable resistance.

AbstractLeucinostatin A ist eine der potentesten antiprotozoischen Verbindungen, die jemals beschrieben wurden, aber bisher war wenig über die Struktur‐Aktivitäts‐Beziehung (SAR) bekannt. Trypanosoma brucei wurde als Protozoen‐Modellorganismus verwendet, um synthetisch modifizierte Derivate zu testen. Dabei wurden die vereinfachten, aber gleichermaßen aktiven Verbindungen 2 (ZHAWOC6025) und 4 (ZHAWOC6027) identifiziert. Anschließend wurden Modifikationen in allen Teilen des Moleküls durchgeführt, um ein besseres SAR‐Verständnis zu erlangen. Die antiprotozoische SAR stimmte mit der SAR in Phospholipid‐Liposomen überein, wobei die Membranintegrität, das Durchlässigkeitsverhalten und die Dynamik untersucht wurden. Die antiprotozoale Wirkung der natürlichen und synthetischen Leucinostatine liegt in der Destabilisierung der inneren Mitochondrienmembran, wie durch Ultrastrukturanalyse, Elektronenmikroskopie und Mitochondrienfärbung gezeigt wurde. Eine subletale Langzeitexposition von T. brucei (200 Passagen) und ein siRNA‐Screening von 12′000 Mutanten zeigten keine Anzeichen einer Resistenzentwicklung gegenüber den synthetischen Derivaten.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

Given the vast number of venomous and poisonous animals, it is surprising that only relatively few animal-derived toxins have been explored and made their way into marketed drugs or are being investigated in ongoing clinical trials. In this review, we highlight marketed drugs deriving from animal toxins as well as ongoing clinical trials and preclinical investigations in the field. We emphasize that more attention should be paid to the rich supply of candidates that nature provides as valuable starting points for addressing serious unmet medical needs.

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure-activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in-depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure-activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long-time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12'000 mutants showed no signs of resistance development to the synthetic derivatives.

Owing to their structural diversity, peptides are a unique source of innovative active ingredients. However, their development has been challenging because of their disadvantageous pharmacokinetic (PK) properties. Over the past decade, many attempts have been made to improve the oral bioavailability of peptide drugs. In this review, we highlight the most recent and promising techniques aimed at the improvement of the oral bioavailability of peptides. The most recent findings will influence future approaches of pharmaceutical companies in the development of new, more efficient, and safer orally delivered peptides.