Correction to "Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity".

[This corrects the article DOI: 10.1021/acsptsci.3c00313.].

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.

Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity.

Cathepsins (Cats) are proteases that mediate the successful entry of SARS-CoV-2 into host cells. We designed and synthesized a tailored series of 21 peptidomimetics and evaluated their inhibitory activity against human cathepsins L, B, and S. Structural diversity was realized by combinations of different C-terminal warhead functions and N-terminal capping groups, while a central Leu-Phe fragment was maintained. Several compounds were identified as promising cathepsin L and S inhibitors with Ki values in the low nanomolar to subnanomolar range, for example, the peptide aldehydes 9a and 9b (9a, 2.67 nM, CatL; 0.455 nM, CatS; 9b, 1.76 nM, CatL; 0.512 nM, CatS). The compounds' inhibitory activity against the main protease of SARS-CoV-2 (Mpro) was additionally investigated. Based on the results at CatL, CatS, and Mpro, selected inhibitors were subjected to investigations of their antiviral activity in cell-based assays. In particular, the peptide nitrile 11e exhibited promising antiviral activity with an EC50 value of 38.4 nM in Calu-3 cells without showing cytotoxicity. High metabolic stability and favorable pharmacokinetic properties make 11e suitable for further preclinical development.

Aerosolized delivery of ESKAPE pathogens for murine pneumonia models.

Murine pneumonia models for ESKAPE pathogens serve to evaluate novel antibacterials or to investigate immunological responses. The majority of published models uses intranasal or to a limited extent the intratracheal instillation to challenge animals. In this study, we propose the aerosol delivery of pathogens using a nebulizer. Aerosol delivery typically results in homogeneous distribution of the inoculum in the lungs because of lower particle size. This is of particular importance when compounds are assessed for their pharmacokinetic and pharmacodynamic (PK/PD) relationships as it allows to conduct several analysis with the same sample material. Moreover, aerosol delivery has the advantage that it mimics the 'natural route' of respiratory infection. In this short and concise study, we show that aerosol delivery of pathogens resulted in a sustained bacterial burden in the neutropenic lung infection model for five pathogens tested, whereas it gave a similar result in immunocompetent mice for three out of five pathogens. Moreover, a substantial bacterial burden in the lungs was already achieved 2 h post inhalation. Hence, this study constitutes a viable alternative for intranasal administration and a refinement of murine pneumonia models for PK/PD assessments of novel antibacterial compounds allowing to study multiple readouts with the same sample material.

Pharmacokinetic and pharmacodynamic evaluation of the atypical tetracyclines chelocardin and amidochelocardin in murine infection models.

IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.

Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Sorangicin A Is Active against Chlamydia in Cell Culture, Explanted Fallopian Tubes, and Topical In Vivo Treatment.

Current treatment of Chlamydia trachomatis using doxycycline and azithromycin introduces detrimental side effects on the host's microbiota. As a potential alternative treatment, the myxobacterial natural product sorangicin A (SorA) blocks the bacterial RNA polymerase. In this study we analyzed the effectiveness of SorA against C. trachomatis in cell culture, and explanted fallopian tubes and systemic and local treatment in mice, providing also pharmacokinetic data on SorA. Potential side effects of SorA on the vaginal and gut microbiome were assessed in mice and against human-derived Lactobacillus species. SorA showed minimal inhibitory concentrations of 80 ng/mL (normoxia) to 120 ng/mL (hypoxia) against C. trachomatis in vitro and was eradicating C. trachomatis at a concentration of 1 µg/mL from fallopian tubes. In vivo, SorA reduced chlamydial shedding by more than 100-fold within the first days of infection by topical application corresponding with vaginal detection of SorA only upon topical treatment, but not after systemic application. SorA changed gut microbial composition during intraperitoneal application only and did neither alter the vaginal microbiota in mice nor affect growth of human-derived lactobacilli. Additional dose escalations and/or pharmaceutical modifications will be needed to optimize application of SorA and to reach sufficient anti-chlamydial activity in vivo.

All Roads Lead to Rome: Enhancing the Probability of Target Attainment with Different Pharmacokinetic/Pharmacodynamic Modelling Approaches.

In light of rising antimicrobial resistance and a decreasing number of antibiotics with novel modes of action, it is of utmost importance to accelerate development of novel treatment options. One aspect of acceleration is to understand pharmacokinetics (PK) and pharmacodynamics (PD) of drugs and to assess the probability of target attainment (PTA). Several in vitro and in vivo methods are deployed to determine these parameters, such as time-kill-curves, hollow-fiber infection models or animal models. However, to date the use of in silico methods to predict PK/PD and PTA is increasing. Since there is not just one way to perform the in silico analysis, we embarked on reviewing for which indications and how PK and PK/PD models as well as PTA analysis has been used to contribute to the understanding of the PK and PD of a drug. Therefore, we examined four recent examples in more detail, namely ceftazidime-avibactam, omadacycline, gepotidacin and zoliflodacin as well as cefiderocol. Whereas the first two compound classes mainly relied on the 'classical' development path and PK/PD was only deployed after approval, cefiderocol highly profited from in silico techniques that led to its approval. Finally, this review shall highlight current developments and possibilities to accelerate drug development, especially for anti-infectives.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Synthesis and Characterization of DOTAM-Based Sideromycins for Bacterial Imaging and Antimicrobial Therapy.

The rise of antimicrobial resistance, especially in Gram-negative bacteria, calls for novel diagnostics and antibiotics. To efficiently penetrate their double-layered cell membrane, we conjugated the potent antibiotics daptomycin, vancomycin, and sorangicin A to catechol siderophores, which are actively internalized by the bacterial iron uptake machinery. LC-MS/MS uptake measurements of sorangicin derivatives verified that the conjugation led to a 100- to 525-fold enhanced uptake into bacteria compared to the free drug. However, the transfer to the cytosol was insufficient, which explains their lack of antibiotic efficacy. Potent antimicrobial effects were observed for the daptomycin conjugate 7 (∼1 µM) against multidrug-resistant Acinetobacter baumannii. A cyanin-7 label aside the daptomycin warhead furnished the theranostic 13 that retained its antibiotic activity and was also able to label ESKAPE bacteria, as demonstrated by microscopy and fluorescence assays. 13 and the cyanin-7 imaging conjugate 14 were stable in human plasma and had low plasma protein binding and cytotoxicity.

Neutralizing the Impact of the Virulence Factor LecA from Pseudomonas aeruginosa on Human Cells with New Glycomimetic Inhibitors.

Bacterial adhesion, biofilm formation and host cell invasion of the ESKAPE pathogen Pseudomonas aeruginosa require the tetravalent lectins LecA and LecB, which are therefore drug targets to fight these infections. Recently, we have reported highly potent divalent galactosides as specific LecA inhibitors. However, they suffered from very low solubility and an intrinsic chemical instability due to two acylhydrazone motifs, which precluded further biological evaluation. Here, we isosterically substituted the acylhydrazones and systematically varied linker identity and length between the two galactosides necessary for LecA binding. The optimized divalent LecA ligands showed improved stability and were up to 1000-fold more soluble. Importantly, these properties now enabled their biological characterization. The lead compound L2 potently inhibited LecA binding to lung epithelial cells, restored wound closure in a scratch assay and reduced the invasiveness of P. aeruginosa into host cells.

Inhibitors of the Elastase LasB for the Treatment of Pseudomonas aeruginosa Lung Infections.

Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa are emerging worldwide as a major threat to human health. Conventional antibiotic monotherapy suffers from rapid resistance development, underlining urgent need for novel treatment concepts. Here, we report on a nontraditional approach to combat P. aeruginosa-derived infections by targeting its main virulence factor, the elastase LasB. We discovered a new chemical class of phosphonates with an outstanding in vitro ADMET and PK profile, auspicious activity both in vitro and in vivo. We established the mode of action through a cocrystal structure of our lead compound with LasB and in several in vitro and ex vivo models. The proof of concept of a combination of our pathoblocker with levofloxacin in a murine neutropenic lung infection model and the reduction of LasB protein levels in blood as a proof of target engagement demonstrate the great potential for use as an adjunctive treatment of lung infections in humans.

Lectin-Targeted Prodrugs Activated by Pseudomonas aeruginosa for Self-Destructive Antibiotic Release.

Chronic Pseudomonas aeruginosa infections are characterized by biofilm formation, a major virulence factor of P. aeruginosa and cause of extensive drug resistance. Fluoroquinolones are effective antibiotics but are linked to severe side effects. The two extracellular P. aeruginosa-specific lectins LecA and LecB are key structural biofilm components and can be exploited for targeted drug delivery. In this work, several fluoroquinolones were conjugated to lectin probes by cleavable peptide linkers to yield lectin-targeted prodrugs. Mechanistically, these conjugates therefore remain non-toxic in the systemic distribution and will be activated to kill only once they have accumulated at the infection site. The synthesized prodrugs proved stable in the presence of host blood plasma and liver metabolism but rapidly released the antibiotic cargo in the presence of P. aeruginosa in a self-destructive manner in vitro. Furthermore, the prodrugs showed good absorption, distribution, metabolism, and elimination (ADME) properties and reduced toxicity in vitro, thus establishing the first lectin-targeted antibiotic prodrugs against P. aeruginosa.

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.

Pharmacokinetics and Pharmacodynamics (PK/PD) of Corallopyronin A against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization's high priority pathogen organism, with an estimated > 100,000 deaths worldwide in 2019. Thus, there is an unmet medical need for novel and resistance-breaking anti-infectives. The natural product Co-rallopyronin A (CorA), currently in preclinical development for filariasis, is efficacious against MRSA in vitro. In this study, we evaluated the pharmacokinetics of CorA after dosing in mice. Furthermore, we determined compound concentrations in target compartments, such as lung, kidney and thigh tissue, using LC-MS/MS. Based on the pharmacokinetic results, we evaluated the pharmacodynamic profile of CorA using the standard neutropenic thigh and lung infection models. We demonstrate that CorA is effective in both standard pharmacodynamic models. In addition to reaching effective levels in the lung and muscle, CorA was detected at high levels in the thigh bone. The data presented herein encourage the further exploration of the additional CorA indications treatment of MRSA- and methicillin-sensitive S. aureus- (MSSA) related infections.

Physiologically Based Pharmacokinetic/Pharmacodynamic Model for the Treatment of Dengue Infections Applied to the Broad Spectrum Antiviral Soraphen A.

While a drug treatment is unavailable, the global incidence of Dengue virus (DENV) infections and its associated severe manifestations continues to rise. We report the construction of the first physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model that predicts viremia levels in relevant target organs based on preclinical data with the broad spectrum antiviral soraphen A (SorA), an inhibitor of the host cell target acetyl-CoA-carboxylase. SorA was highly effective against DENV in vitro (EC50 = 4.7 nM) and showed in vivo efficacy by inducing a significant reduction of viral load in the spleen and liver of IFNAR-/- mice infected with DENV-2. PBPK/PD predictions for SorA matched well with the experimental infection data. Transfer to a human PBPK/PD model for DENV to mimic a clinical scenario predicted a reduction in viremia by more than one log10 unit for an intravenous infusion regimen of SorA. The PBPK/PD model is applicable to any DENV drug lead and, thus, represents a valuable tool to accelerate and facilitate DENV drug discovery and development.

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors.

Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in 'real-life' human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at -20 °C and room temperature. Whereas all metabolites were stable at -20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.