Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Discovery of N-ß-l-Fucosyl Amides as High-Affinity Ligands for the Pseudomonas aeruginosa Lectin LecB.

The Gram-negative pathogen Pseudomonas aeruginosa causes severe infections mainly in immunocompromised or cystic fibrosis patients and is able to resist antimicrobial treatments. The extracellular lectin LecB plays a key role in bacterial adhesion to the host and biofilm formation. For the inhibition of LecB, we designed and synthesized a set of fucosyl amides, sulfonamides, and thiourea derivatives. Then, we analyzed their binding to LecB in competitive and direct binding assays. We identified ß-fucosyl amides as unprecedented high-affinity ligands in the two-digit nanomolar range. X-ray crystallography of one α- and one ß-anomer of N-fucosyl amides in complex with LecB revealed the interactions responsible for the high affinity of the ß-anomer at atomic level. Further, the molecules showed good stability in murine and human blood plasma and hepatic metabolism, providing a basis for future development into antibacterial drugs.

Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (Mpro, 3CLpro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. Mpro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 Mpro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC50 of 120 nM against the Mpro and EC50 values of 0.8-3.4 µM for antiviral activity in different cell types. Crystal structures have been elucidated for the Mpro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.

Identification of Antimotilins, Novel Inhibitors of Helicobacter pylori Flagellar Motility That Inhibit Stomach Colonization in a Mouse Model.

New treatment options against the widespread cancerogenic gastric pathogen Helicobacter pylori are urgently needed. We describe a novel screening procedure for inhibitors of H. pylori flagellar biosynthesis. The assay is based on a flaA flagellin gene-luciferase reporter fusion in H. pylori and was amenable to multi-well screening formats with an excellent Z factor. We screened various compound libraries to identify virulence blockers ("antimotilins") that inhibit H. pylori motility or the flagellar type III secretion apparatus. We identified compounds that either inhibit both motility and the bacterial viability, or the flagellar system only, without negatively affecting bacterial growth. Novel anti-virulence compounds which suppressed flagellar biosynthesis in H. pylori were active on pure H. pylori cultures in vitro and partially suppressed motility directly, reduced flagellin transcript and flagellin protein amounts. We performed a proof-of-principle treatment study in a mouse model of chronic H. pylori infection and demonstrated a significant effect on H. pylori colonization for one antimotilin termed Active2 even as a monotherapy. The diversity of the intestinal microbiota was not significantly affected by Active2. In conclusion, the novel antimotilins active against motility and flagellar assembly bear promise to complement commonly used antibiotic-based combination therapies for treating and eradicating H. pylori infections. IMPORTANCE Helicobacter pylori is one of the most prevalent bacterial pathogens, inflicting hundreds of thousands of peptic ulcers and gastric cancers to patients every year. Antibacterial treatment of H. pylori is complicated due to the need of combining multiple antibiotics, entailing serious side effects and increasing selection for antibiotic resistance. Here, we aimed to explore novel nonantibiotic approaches to H. pylori treatment. We selected an antimotility approach since flagellar motility is essential for H. pylori colonization. We developed a screening system for inhibitors of H. pylori motility and flagellar assembly, and identified numerous novel antibacterial and anti-motility compounds (antimotilins). Selected compounds were further characterized, and one was evaluated in a preclinical therapy study in mice. The antimotilin compound showed a good efficacy to reduce bacterial colonization in the model, such that the antimotilin approach bears promise to be further developed into a therapy against H. pylori infection in humans.

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound's polypharmacology.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates.

The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines (e.g. 16R-aminoratjadone 16 with IC50 = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC50 = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy.

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 µg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 µg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.

Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas aeruginosa.

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.

Disorazoles Block Group A Streptococcal Invasion into Epithelial Cells Via Interference with the Host Factor Ezrin.

Bacterial pathogens use invasion into human cells as a strategy to escape not only the host's immune response, but also anti-bacterial treatment. This often leads to persistence and enables reinitiation of the infection process at a later time point. Here, we show that a family of myxobacterial metabolites, disorazoles, block invasion of group A Streptococcus (GAS) into human epithelial cells. Mechanistically, disorazoles target ezrin, a host protein involved in linking microfilaments to the membrane, and affect invasion most likely by interfering with dynamic phosphorylation of ezrin. Overall, our study suggests ezrin as a new factor in two different GAS invasion pathways, independent of the already known CD44 pathway, and that disorazoles are promising "pathoblocker" compounds aimed at this additional invasion mechanism.