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Efficient identification of drug mechanisms of action remains a challenge. Computational docking approaches have been widely used to predict drug binding targets; yet, such approaches depend on existing protein structures, and accurate structural predictions have only recently become available from AlphaFold2. Here, we combine AlphaFold2 with molecular docking simulations to predict protein‐ligand interactions between 296 proteins spanning Escherichia coli 's essential proteome, and 218 active antibacterial compounds and 100 inactive compounds, respectively, pointing to widespread compound and protein promiscuity. We benchmark model performance by measuring enzymatic activity for 12 essential proteins treated with each antibacterial compound. We confirm extensive promiscuity, but find that the average area under the receiver operating characteristic curve (auROC) is 0.48, indicating weak model performance. We demonstrate that rescoring of docking poses using machine learning‐based approaches improves model performance, resulting in average auROCs as large as 0.63, and that ensembles of rescoring functions improve prediction accuracy and the ratio of true‐positive rate to false‐positive rate. This work indicates that advances in modeling protein‐ligand interactions, particularly using machine learning‐based approaches, are needed to better harness AlphaFold2 for drug discovery. Synopsis Assessing molecular docking simulations based on AlphaFold2‐predicted structures with high‐throughput measurements of protein‐ligand interactions reveals weak model performance. Machine learning‐based approaches improve performance and better harness AlphaFold2 for drug discovery. AlphaFold2‐based molecular docking predictions for 296 Escherichia coli proteins, 218 active antibacterial compounds and 100 inactive compounds predict widespread promiscuity and similar distributions of binding affinities between active and inactive compounds. Quantitative enzymatic inhibition assays for 12 essential E. coli proteins treated with each of the 218 antibacterial compounds confirm extensive promiscuity. The enzymatic inhibition dataset reveals that the performance of the molecular docking model is weak. Rescoring of docking poses using machine learning‐based scoring functions improves model performance. Graphical Abstract Assessing molecular docking simulations based on AlphaFold2‐predicted structures with high‐throughput measurements of protein‐ligand interactions reveals weak model performance. Machine learning‐based approaches improve performance and better harness AlphaFold2 for drug discovery.

Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics. Graphical Abstract Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease‐associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full‐length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α‐secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α‐secretase‐mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho‐SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid β‐peptide in vitro . In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9‐bound. Moreover, in a mouse model for Alzheimer's disease‐related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease‐associated state. Synopsis This study describes the discovery and characterization of a novel TREM2 antibody, which induces protective microglial functions and provides a basis for the development of human antibodies with a similar mechanistic profile for treatment of Alzheimer's disease. An antibody directed to the stalk region of TREM2 prevents its shedding and increases cell autonomous signaling. Addition of this TREM2 antibody to myeloid cells in vitro stimulates phagocytosis, and improves cell survival. TREM2 antibody treatment increases TREM2 expression on brain microglia, decreases homeostatic markers and reduces amyloid plaque pathology in a mouse model of Alzheimer's disease. Antibody mediated stimulation of TREM2 signaling may be efficacious in Alzheimer's disease as well as other neurodegenerative disorders and obesity‐associated metabolic syndromes. Graphical Abstract This study describes the discovery and characterization of a novel TREM2 antibody, which induces protective microglial functions and provides a basis for the development of human antibodies with a similar mechanistic profile for treatment of Alzheimer's disease.

High‐throughput (HT) screening drug discovery, during which thousands or millions of compounds are screened, remains the key methodology for identifying active chemical matter in early drug discovery pipelines. Recent technological developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabelled biomolecules in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label‐free MS assays are often cheaper, faster, and more physiologically relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label‐free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compounds in cell lines, and even primary cells. Finally, we will give an outlook on how technological advances will increase the future use and the capabilities of mass spectrometry in drug discovery. Graphical Abstract This Review summarizes advantages and disadvantages of high‐throughput mass spectrometry techniques used in drug discovery and discusses how technological advances could increase the capabilities of mass spectrometry in drug discovery in the future.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca 2+ ‐permeable endolysosomal two‐pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann–Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in‐vivo . Synopsis Mutations in proteins of the endolysosomal machinery such as lysosomal enzymes or lysosomal membrane proteins often result in severe neurodegenerative disease, including mucolipidosis type IV, Niemann Pick type C1, or Batten disease. Small molecule activation of the endolysosomal two‐pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with lysosomal storage diseases (LSDs), such as the accumulation of cholesterol, lactosylceramide, or lipofuscin, or the formation of abnormal vacuoles. Rescue effects of the TPC2 agonist are demonstrated in LSD patient fibroblasts and neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. Mechanistically, the fully preserved capability of TPC2 to promote lysosomal exocytosis and autophagy in these diseases is demonstrated, suggesting rescue effects by clearing intracellular debris. Expression patterns of TPC2 in the brain are examined using a newly engineered TPC2‐GFP reporter mouse model, complemented by qPCR analyses of human and mouse brain samples. In vivo efficacy of the PI(3,5)P2‐mimetic TPC2 agonist TPC2‐A1‐P is shown, restoring pathological hallmarks in a mouse model of MLIV. Graphical Abstract Mutations in proteins of the endolysosomal machinery such as lysosomal enzymes or lysosomal membrane proteins often result in severe neurodegenerative disease, including mucolipidosis type IV, Niemann–Pick type C1, or Batten disease.

The recent years saw the advent of promising preclinical strategies that combat the devastating effects of a spinal cord injury (SCI) that are progressing towards clinical trials. However, individually, these treatments produce only modest levels of recovery in animal models of SCI that could hamper their implementation into therapeutic strategies in spinal cord injured humans. Combinational strategies have demonstrated greater beneficial outcomes than their individual components alone by addressing multiple aspects of SCI pathology. Clinical trial designs in the future will eventually also need to align with this notion. The scenario will become increasingly complex as this happens and conversations between basic researchers and clinicians are required to ensure accurate study designs and functional readouts. Graphical Abstract This review is comprehensive and timely, and provides to the readers a balanced view of research and clinical findings in spinal cord injury.

Treatment options for COVID‐19, caused by SARS‐CoV‐2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS‐CoV‐2–host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS‐CoV‐2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP‐MS) and the complementary proximity‐based labeling MS method (BioID‐MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS‐CoV‐2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image‐based drug screen with infectious SARS‐CoV‐2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein–protein interactions. Synopsis A large‐scale proteomics study identifies critical host proteins for SARS‐CoV‐2 processing. Proteins from these core subnetworks are used for drug repurposing analyses, indicating drugs with antiviral effects. A large‐scale proteomics study identifies 4,781 unique high‐confidence virus‐host protein‐protein interactions (PPIs) using 29 viral ORFs, and 4,362 unique PPIs for 18 suggested receptors/proteases/cofactors for SARS‐CoV‐2. The characterization of 693 hub proteins that connect viral baits and host baits via a dense network reveals critical host pathways used for viral replication. 59 compounds are prioritized that could be repurposed for 15 host protein targets used by the SARS‐CoV‐2 during the infection. Ten candidate drugs are validated using an image‐based drug screen assay, six of them demonstrating antiviral effects. Graphical Abstract A large‐scale proteomics study identifies critical host proteins for SARS‐CoV‐2 processing. Proteins from these core subnetworks are used for drug repurposing analyses, indicating drugs with antiviral effects.

Initially used as antimalarial drugs, hydroxychloroquine (HCQ) and, to a lesser extent, chloroquine (CQ) are currently being used to treat several diseases. Due to its cost‐effectiveness, safety and efficacy, HCQ is especially used in rheumatic autoimmune disorders (RADs), such as systemic lupus erythematosus, primary Sjögren's syndrome and rheumatoid arthritis. Despite this widespread use in the clinic, HCQ molecular modes of action are still not completely understood. By influencing several cellular pathways through different mechanisms, CQ and HCQ inhibit multiple endolysosomal functions, including autophagy, as well as endosomal Toll‐like receptor activation and calcium signalling. These effects alter several aspects of the immune system with the synergistic consequence of reducing pro‐inflammatory cytokine production and release, one of the most marked symptoms of RADs. Here, we review the current knowledge on the molecular modes of action of these drugs and the circumstances under which they trigger side effects. This is of particular importance as the therapeutic use of HCQ is expanding beyond the treatment of malaria and RADs. Graphical Abstract Hydroxychloroquine has been heavily discussed in the context of COVID19, but this anti‐malarial drug is primarily used in rheumatic autoimmune disorders (RADs). This comprehensive review recapitulates our knowledge on the mode of action of this drug in RADs, and on its potential applications and side effects.

A small but significant proportion of COVID‐19 patients develop life‐threatening cytokine storm. We have developed a new anti‐inflammatory drug, EXO‐CD24, a combination of an immune checkpoint (CD24) and a delivery platform (exosomes). CD24 inhibits the NF‐kB pathway and the production of cytokines/chemokines. EXO‐CD24 discriminates damage‐from pathogen‐associated molecular patterns (DAMPs and PAMPs) therefore does not interfere with viral clearance. EXO‐CD24 was produced and purified from CD24‐expressing 293‐TREx™ cells. Exosomes displaying murine CD24 (mCD24) were also created. EXO‐CD24/mCD24 were characterized and examined, for safety and efficacy, in vitro and in vivo . In a phase Ib/IIa study, 35 patients with moderate–high severity COVID‐19 were recruited and given escalating doses, 10 8 –10 10 , of EXO‐CD24 by inhalation, QD, for 5 days. No adverse events related to the drug were observed up to 443–575 days. EXO‐CD24 effectively reduced inflammatory markers and cytokine/chemokine, although randomized studies are required. EXO‐CD24 may be a treatment strategy to suppress the hyper‐inflammatory response in the lungs of COVID‐19 patients and further serve as a therapeutic platform for other pulmonary and systemic diseases characterized by cytokine storm. Synopsis In 5% of COVID‐19 patients, 5–10 days from disease onset, there is rapid clinical deterioration due to the cytokine storm with no effective therapy. EXO‐CD24 is the new immunomodulator with promising efficacy without interfering with pathogen clearance. EXO‐CD24 is a novel platform that helps normalize immune activity. EXO‐CD24 may represent a new therapeutic opportunity to overcome the devastating effect of COVID‐19 and beyond. EXO‐CD24 leads to inhibition of tissue injury‐driven inflammation without interfering with pathogen‐induced immune activation. EXO‐CD24 is a targeted innovative technology, based on CD24‐enriched exosomes, delivered directly to the lungs to suppress the cytokine storm, and has broad applicability. Graphical Abstract In 5% of COVID‐19 patients, 5–10 days from disease onset, there is rapid clinical deterioration due to the cytokine storm with no effective therapy. EXO‐CD24 is the new immunomodulator with promising efficacy without interfering with pathogen clearance.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID‐19 infection via proposed anti‐cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID‐19 infection. We validated the AI‐predicted biochemical inhibitory effects of baricitinib on human numb‐associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Collectively, these data support further evaluation of the anti‐cytokine and anti‐viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID‐19 patients. Synopsis This study provides biochemical and cellular evidence confirming artificial intelligence (AI)‐predictions focused on anti‐cytokine signaling and potential anti‐viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID‐19 patients. Baricitinib, an oral Janus kinase (JAK)1/JAK2 inhibitor used to treat rheumatoid arthritis, was hypothesised using AI to be useful in COVID‐19. Baricitinib‐mediated inhibition of numb associated kinases utilized by SARS‐CoV‐2 for its propagation, led to reduced viral infectivity in primary liver spheroids. Baricitinib reduces levels of cytokines implicated in COVID‐19 and inhibits their signaling. In patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Graphical Abstract This study provides biochemical and cellular evidence confirming artificial intelligence (AI)‐predictions focused on anti‐cytokine signaling and potential anti‐viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID‐19 patients.