Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets.

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs' host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

An organometallic hybrid antibiotic of metronidazole with a Gold(I) N-Heterocyclic Carbene overcomes metronidazole resistance in Clostridioides difficile.

Antimicrobial resistance (AMR) has been emerging as a major global health threat and calls for the development of novel drug candidates. Metal complexes have been demonstrating high efficiency as antibacterial agents that differ substantially from the established types of antibiotics in their chemical structures and their mechanism of action. One strategy to exploit this potential is the design of metal-based hybrid organometallics that consist of an established antibiotic and a metal-based warhead that contributes an additional mechanism of action different from that of the parent antibiotic. In this communication, we describe the organometallic hybrid antibiotic 2c, in which the drug metronidazole is connected to a gold(I) N-heterocyclic carbene warhead that inhibits bacterial thioredoxin reductase (TrxR). Metronidazole can be used for the treatment with the obligatory anaerobic pathogen Clostridioides difficile (C. difficile), however, resistance to the drug hampers its clinical success. The gold organometallic conjugate 2c was an efficient inhibitor of TrxR and it was inactive or showed only minor effects against eucaryotic cells and bacteria grown under aerobic conditions. In contrast, a strong antibacterial effect was observed against both metronidazole-sensitive and -resistant strains of C. difficile. This report presents a proof-of-concept that the design of metal-based hybrid antibiotics can be a viable approach to efficiently tackle AMR.

Detection of bornavirus-reactive antibodies and BoDV-1 RNA only in encephalitis patients from virus endemic areas: a comparative serological and molecular sensitivity, specificity, predictive value, and disease duration correlation study.

PURPOSE: Human Borna disease virus (BoDV-1) encephalitis is an emerging disease in Germany. This study investigates the spectrum of human BoDV-1 infection, characterizes anti-BoDV-1-antibodies and kinetics, and compares laboratory test performances. METHODS: Three hundred four encephalitis cases, 308 nation-wide neuropsychiatric conditions, 127 well-defined psychiatric cases from Borna disease-endemic areas, and 20 persons with contact to BoDV-1 encephalitis patients or animals were tested for BoDV-1 infections by serology and PCR. RESULTS: BoDV-1 infections were only found in encephalitis patients with residence in, or recent travel to, virus-endemic areas. Antibodies were detected as early as 12 days after symptom onset. Serum antibody levels correlated with disease duration. Serology was ordered after 50% of the disease duration had elapsed, reflecting low awareness. BoDV-1-antibodies were of IgG1 subclass, and the epitope on BoDV-1 antigens was determined. Specificity of the indirect immunofluorescence antibody test (IFAT) and lineblot (LB) from serum and cerebrospinal fluid (CSF), as well as PCR testing from CSF, was 100%. Sensitivity, depending on first or all samples, reached 75-86% in serum and 92-94% in CSF for the IFAT, and 33-57% in serum and 18-24% in CSF for the LB. Sensitivity for PCR in CSF was 25-67%. Positive predictive values were 100% each, while negative predictive values were 99% (IFAT), 91-97% (LB), and 90% (PCR). CONCLUSIONS: There is no hint that BoDV-1 causes other diseases than encephalitis in humans. Awareness has to be increased in virus-endemic areas. Tests are robust but lack sensitivity. Detection of IgG1 against specific peptides may facilitate diagnosis. Screening of healthy individuals is likely not beneficial.

Depicting the Chemical Diversity of Bioactive Meroterpenoids Produced by the Largest Organism on Earth.

In this investigation, we explored the diversity of melleolide-type meroterpenoids produced by Armillaria ostoyae, one of the largest and oldest organisms on Earth, using extracts from liquid and solid fermentation media. The study unveiled three unprecedented dimeric bismelleolides and three novel fatty-acid-substituted congeners, along with 11 new and 21 known derivatives. The structures were elucidated by 1D and 2D NMR spectroscopy and HRESI-MS, and ROESY spectral analysis for relative configurations. Absolute configurations were determined from crystal structures and through ECD spectra comparison. A compound library of melleolide-type meroterpenoids facilitated metabolomics-wide associations, revealing production patterns under different culture conditions. The library enabled assessments of antimicrobial and cytotoxic activities, revealing that the Δ2,4 double bond is not crucial for antifungal activity. Cytotoxicity was linked to the presence of an aldehyde at C1, but lost with hydroxylation at C13. Chemoinformatic analyses demonstrated the intricate interplay of chemical modifications on biological properties. This study marks the first systematic exploration of Armillaria spp. meroterpenoid diversity by MS-based untargeted metabolomics, offering insight into structure-activity relationships through innovative chemoinformatics.

Establishment of an In Bacterio Assay for the Assessment of Carbon Storage Regulator A (CsrA) Inhibitors.

Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.

Synthesis, biological evaluation, and molecular docking studies of aldotetronic acid-based LpxC inhibitors.

In order to develop novel inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the UDP binding site of the enzyme, a series of aldotetronic acid-based hydroxamic acids was accessed in chiral pool syntheses starting from 4,6-O-benzylidene-d-glucose and l-arabinitol. The synthesized hydroxamic acids were tested for LpxC inhibitory activity in vitro, revealing benzyl ether 17a ((2S,3S)-4-(benzyloxy)-N,3-dihydroxy-2-[(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)oxy]butanamide) as the most potent LpxC inhibitor. This compound was additionally tested for antibacterial activity against a panel of clinically relevant Gram-negative bacteria, bacterial uptake, and susceptibility to efflux pumps. Molecular docking studies were performed to rationalize the observed structure-activity relationships.

A New Way to 2,3,4-Trisubstituted Benzo[h]quinolines: Synthesis, Consecutive Reactions and Cellular Activities †.

The reaction of mercaptoacetic acid esters with pentachloro-2-nitro-1,3-butadiene provides the appropriate precursors for the synthesis of 2,3,4-trisubstituted benzo[h]quinolines. These heterocycles are easily accessible via a single-step reaction with naphthalen-1-amine or anthracen-1-amine as the precursor. Due to the steric bulk and high electron density ring, the ring closure of benzo[h]quinolines takes place exclusively. Such highly substituted annelated pyridine systems can be modified in subsequent, selective reactions to build up new N-heterocycles with promising microbiological properties. The antibacterial and antiproliferative assays against four mammalian cell lines demonstrate that some of the sulfur-substituted benzo[h]quinoline analogs display potent phenotypic bioactivities in the single-digit micromolar range.

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, 55 Fe uptake assays, and LC-MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.

Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids.

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyrase-inhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono- as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbA-mediated inactivation was induced. This provides a proof-of-principle that resistance towards photoswitchable antibiotics can be optically controlled.

Masked Amino Trimethyl Lock (H2 N-TML) Systems: New Molecular Entities for the Development of Turn-On Fluorophores and Their Application in Hydrogen Sulfide (H2 S) Imaging in Human Cells.

Masked trimethyl lock (TML) systems as molecular moieties enabling the bioresponsive release of compounds or dyes in a controlled temporal and spatial manner have been widely applied for the development of drug conjugates, prodrugs or molecular imaging tools. Herein, we report the development of a novel amino trimethyl lock (H2 N-TML) system as an auto-immolative molecular entity for the release of fluorophores. We designed Cou-TML-N3 and MURh-TML-N3 , two azide-masked turn-on fluorophores. The latter was demonstrated to selectively release fluorescent MURh in the presence of physiological concentrations of the redox-signaling molecule H2 S in vitro and was successfully applied to image H2 S in human cells.

A Metabolomics-Based Toolbox to Assess and Compare the Metabolic Potential of Unexplored, Difficult-to-Grow Bacteria.

Novel high-throughput cultivation techniques create a demand to pre-select strains for in-depth follow-up studies. We report a workflow to identify promising producers of novel natural products by systematically characterizing their metabolomes. For this purpose, 60 strains from four phyla (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) comprising 16 novel species and six novel genera were cultivated from marine and terrestrial sources. Their cellular metabolomes were recorded by LC-MS/MS; data analysis comprised databases MS/MS matching, in silico compound assignment, and GNPS-based molecular networking. Overall, 1052 different molecules were identified from 6418 features, among them were unusual metabolites such as 4-methoxychalcone. Only a minor portion of the 755 features were found in all phyla, while the majority occurred in a single phylogroup or even in a single strain. Metabolomic methods enabled the recognition of highly talented strains such as AEG42_45, which had 107 unique features, among which a family of 28 potentially novel and related compounds according to MS/MS similarities. In summary, we propose that high-throughput cultivation and isolation of bacteria in combination with the presented systematic and unbiased metabolome analysis workflow is a promising approach to capture and assess the enormous metabolic potential of previously uncultured bacteria.

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the Mpro. Starting from crystal structures of the Mpro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the Mpro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC50 of 13 nM versus the recombinant Mpro, and similar potency was observed for its P1' N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 Mpro inhibition, we also explored the activity of MG-78 against the Mpro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 Mpro), moderate (1.45 µM, Coxsackievirus 3Cpro), and relatively poor (6.7 µM, enterovirus A71 3Cpro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus Mpros but further optimization would be needed to target enterovirus 3Cpros efficiently.

Enzyme-Activated, Chemiluminescent Siderophore-Dioxetane Probes Enable the Selective and Highly Sensitive Detection of Bacterial Pathogens.

The sensitive detection of bacterial infections is a prerequisite for their successful treatment. The use of a chemiluminescent readout was so far hampered by an insufficient probe enrichment at the pathogens. We coupled siderophore moieties, that harness the unique iron transport system of bacteria, with enzyme-activatable dioxetanes and obtained seven trifunctional probes with high signal-to-background ratios (S/B=426-859). Conjugates with efficient iron transport capability into bacteria were identified through a growth recovery assay. All ESKAPE pathogens were labelled brightly by desferrioxamine conjugates, while catechols were weaker due to self-quenching. Bacteria could also be detected inside lung epithelial cells. The best probe 8 detected 9.1×103  CFU mL-1 of S. aureus and 5.0×104  CFU mL-1 of P. aeruginosa, while the analogous fluorescent probe 10 was 205-305fold less sensitive. This qualifies siderophore dioxetane probes for the selective and sensitive detection of bacteria.

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.

Kaposi's sarcoma-associated herpesvirus vIRF2 protein utilizes an IFN-dependent pathway to regulate viral early gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.

Antibacterial activity of xylose-derived LpxC inhibitors - Synthesis, biological evaluation and molecular docking studies.

LpxC inhibitors represent a promising class of novel antibiotics selectively combating Gram-negative bacteria. In chiral pool syntheses starting from D- and L-xylose, a series of four 2r,3c,4t-configured C-furanosidic LpxC inhibitors was obtained. The synthesized hydroxamic acids were tested for antibacterial and LpxC inhibitory activity, the acquired biological data were compared with those of previously synthesized C-furanosides, and molecular docking studies were performed to rationalize the observed structure-activity relationships. Additionally, bacterial uptake and susceptibility to efflux pump systems were investigated for the most promising stereoisomers.

Selective Bacterial Targeting and Infection-Triggered Release of Antibiotic Colistin Conjugates.

In order to render potent, but toxic antibiotics more selective, we have explored a novel conjugation strategy that includes drug accumulation followed by infection-triggered release of the drug. Bacterial targeting was achieved using a modified fragment of the human antimicrobial peptide ubiquicidin, as demonstrated by fluorophore-tagged variants. To limit the release of the effector colistin only to infection-related situations, we introduced a linker that was cleaved by neutrophil elastase (NE), an enzyme secreted by neutrophil granulocytes at infection sites. The linker carried an optimized sequence of amino acids that was required to assure sufficient cleavage efficiency. The antibacterial activity of five regioisomeric conjugates prepared by total synthesis was masked, but was released upon exposure to recombinant NE when the linker was attached to amino acids at the 1- or the 3-position of colistin. A proof-of-concept was achieved in co-cultures of primary human neutrophils and Escherichia coli that induced the secretion of NE, the release of free colistin, and an antibacterial efficacy that was equal to that of free colistin.

Natural products targeting the elongation phase of eukaryotic protein biosynthesis.

Covering: 2000 to 2020 The translation of mRNA into proteins is a precisely regulated, complex process that can be divided into three main stages, i.e. initiation, elongation, termination, and recycling. This contribution is intended to highlight how natural products interfere with the elongation phase of eukaryotic protein biosynthesis. Cycloheximide, isolated from Streptomyces griseus, has long been the prototype inhibitor of eukaryotic translation elongation. In the last three decades, a variety of natural products from different origins were discovered to also address the elongation step in different manners, including interference with the elongation factors eEF1 and eEF2 as well as binding to A-, P- or E-sites of the ribosome itself. Recent advances in the crystallization of the ribosomal machinery together with natural product inhibitors allowed characterizing similarities as well as differences in their mode of action. Since aberrations in protein synthesis are commonly observed in tumors, and malfunction or overexpression of translation factors can cause cellular transformation, the protein synthesis machinery has been realized as an attractive target for anticancer drugs. The therapeutic use of the first natural products that reached market approval, plitidepsin (Aplidin®) and homoharringtonine (Synribo®), will be introduced. In addition, we will highlight two other potential indications for translation elongation inhibitors, i.e. viral infections and genetic disorders caused by premature termination of translation.

Filovirus Antiviral Activity of Cationic Amphiphilic Drugs Is Associated with Lipophilicity and Ability To Induce Phospholipidosis.

Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood. We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition. We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analyzed correlation of antiviral activity with four chemical properties: pKa, hydrophobicity (octanol/water partitioning coefficient; ClogP), molecular weight, and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structures (analogues) to those of strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall, 11 out of 45 (24%) CADs inhibited MARVpp by 40% or more. The strongest antiviral compounds were dronedarone, triparanol, and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP > 4), and DIPL. Moreover, pKa and intramolecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity but to a lesser extent. We also showed that in contrast to analogues, derivatives had antiviral activity similar to that of the seed compound dronedarone. Overall, one-quarter of CADs inhibit MARVpp entry in vitro, and antiviral activity of CADs mostly relies on their hydrophobicity yet is promoted by the individual structure.

CluMSID: an R package for similarity-based clustering of tandem mass spectra to aid feature annotation in metabolomics.

SUMMARY: Compound identification is one of the most eminent challenges in the untargeted analysis of complex mixtures of small molecules by mass spectrometry. Similarity of tandem mass spectra can provide valuable information on putative structural similarities between known and unknown analytes and hence aids feature identification in the bioanalytical sciences. We have developed CluMSID (Clustering of MS2 spectra for metabolite identification), an R package that enables researchers to make use of tandem mass spectra and neutral loss pattern similarities as a part of their metabolite annotation workflow. CluMSID offers functions for all analysis steps from import of raw data to data mining by unsupervised multivariate methods along with respective (interactive) visualizations. A detailed tutorial with example data is provided as supplementary information. AVAILABILITY AND IMPLEMENTATION: CluMSID is available as R package from https://github.com/tdepke/CluMSID/and from https://bioconductor.org/packages/CluMSID/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.