Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

Antidepressant-like Effect of the Eburnamine-Type Molecule Vindeburnol in Rat and Mouse Models of Ultrasound-Induced Depression.

Vindeburnol (VIND, RU24722, BC19), a synthetic molecule derived from the eburnamine-vincamine alkaloid group, has many neuropsychopharmacological effects, but its antidepressant-like effects are poorly understood and have only been described in a few patents. To reliably estimate this effect, vindeburnol was studied in a model of long-term variable-frequency ultrasound (US) exposure at 20-45 kHz in male Wistar rats and BALB/c mice. Vindeburnol was administered chronically for 21 days against a background of simultaneous ultrasound exposure at a dose of 20 mg/kg intraperitoneally (IP). Using four behavioral tests, the sucrose preference test (SPT), the social interaction test (SIT), the open field test (OFT), and the forced swimming test (FST), we found that the treatment with the compound diminished depression-like symptoms in mice and rats. The compound restored the ultrasound-related reduced sucrose consumption to control levels and increased social interaction time in mice and rats compared with those in ultrasound-exposed animals. Vindeburnol showed contraversive results of horizontal and vertical activity in both species and generally did not increase locomotor activity. At the same time, the compound showed a specific effect in the FST, significantly reducing the immobility time. Moreover, we found an increase in norepinephrine, dopamine, and its metabolite levels in the brainstem, as well as an increase in dopamine, 3-methoxytyramine, and 3,4-dihydroxyphenylacetic acid levels in the striatum. We also observed a statistically significant increase in tyrosine hydroxylase (TH) levels in the region containing the locus coeruleus (LC). We suggest that using its distinct chemical structure and pharmacological activity as a starting point could boost antidepressant drug discovery.

Molecular mechanism of rhinovirus escape from the Pyrazolo[3,4-d]pyrimidine capsid-binding inhibitor OBR-5-340 via mutations distant from the binding pocket: Derivatives that brake resistance.

Rhinoviruses (RVs) cause the common cold. Attempts at discovering small molecule inhibitors have mainly concentrated on compounds supplanting the medium chain fatty acids residing in the sixty icosahedral symmetry-related hydrophobic pockets of the viral capsid of the Rhinovirus-A and -B species. High-affinity binding to these pockets stabilizes the capsid against structural changes necessary for the release of the ss(+) RNA genome into the cytosol of the host cell. However, single-point mutations may abolish this binding. RV-B5 is one of several RVs that are naturally resistant against the well-established antiviral agent pleconaril. However, RV-B5 is strongly inhibited by the pyrazolopyrimidine OBR-5-340. Here, we report on isolation and characterization of RV-B5 mutants escaping OBR-5-340 inhibition and show that substitution of amino acid residues not only within the binding pocket but also remote from the binding pocket hamper inhibition. Molecular dynamics network analysis revealed that strong inhibition occurs when an ensemble of several sequence stretches of the capsid proteins enveloping OBR-5-340 move together with OBR-5-340. Mutations abrogating this dynamic, regardless of whether being localized within the binding pocket or distant from it result in escape from inhibition. Pyrazolo [3,4-d]pyrimidine derivatives overcoming OBR-5-340 escape of various RV-B5 mutants were identified. Our work contributes to the understanding of the properties of capsid-binding inhibitors necessary for potent and broad-spectrum inhibition of RVs.

Benzothiozinone derivatives with anti-tubercular Activity-Further side chain investigation.

A series of novel benzothiozinone (BTZ) derivatives were designed, prepared and evaluated for antituberculosis activity. Specifically, the BTZ pharmacophore is retained and the previous heterocyclic ring linker is replaced by alkynyl or vinyl linker, the resulting compounds displayed about 5-fold improved antimycobacterial activity. We further revealed that the linker attached tail group affects the compound metabolic stability, potency and other drug like properties. This work led to the discovery of two compounds (A1 and A11) with acceptable low MICs and improved metabolic stability. The representative compound A11 demonstrated bactericidal efficacy in an acute TB infection mouse model.

Determination of alkaloid-inspired molecule vindeburnol in rabbit plasma by UPLC-HRMS and its application to pharmacokinetic studies and preliminary metabolite identification.

The eburnamine-vincamine alkaloids exhibit a range of pharmacological activities. There is a limited understanding of the pharmacokinetics and pharmacodynamics of vindeburnol, a synthetic derivative of this chemical class of alkaloids. A fast and reliable UPLC-HRMS method was developed and validated to quantify vindeburnol in Soviet Chinchilla rabbit plasma from pharmacokinetics studies. An ultra-performance liquid chromatography system equipped with a Waters Acquity UPLC HSS T3 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and acetonitrile. An Impact II QqTOF high-resolution mass spectrometer equipped with an Apollo II electrospray ionization source was used for analysis in positive mode; the ions [M+H]+m/z 269.1648 ± 0.003 and m/z 351.2067 ± 0.003 were monitored for vindeburnol and internal standard (vinpocetine), respectively. Preliminary metabolite profiling was also performed, and the pharmacokinetics of the identified metabolites were evaluated. The mean retention times for vindeburnol and vinpocetine were 2.0 and 3.5 min. The UPLC-HRMS method was validated with accuracy and precision within the 15% acceptance limit (8.2% and 11.0%, respectively). The mean extraction recovery value of vindeburnol from rabbit plasma was 77%. Pharmacokinetic evaluation of vindeburnol revealed that the compound is distributed rapidly with a short elimination half-life. Vindeburnol undergoes extensive first-pass metabolism and is metabolized into hydroxyvindeburnol and vindeburnol glucuronide.

1-Sulfonyl-3-amino-1H-1,2,4-triazoles as Yellow Fever Virus Inhibitors: Synthesis and Structure-Activity Relationship.

Yellow fever virus (YFV) transmitted by infected mosquitoes causes an acute viral disease for which there are no approved small-molecule therapeutics. Our recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. We report that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication. As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 and RCB16007 demonstrate selectivity for YFV over related viruses, with only RCB16007 showing some inhibition of the West Nile virus (EC50 7.9 µM, CC50 17 µM, SI 2.2). We also describe the absorption, distribution, metabolism, and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice. This compound had previously been shown to not inhibit hERG, and we now describe that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding, and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T1/2 of 3.4 h and Cmax of 1190 ng/mL, suggesting good availability and stability. We now propose that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class may be prioritized for in vivo efficacy testing against YFV.

Antiviral Evaluation of Dispirotripiperazines against Hepatitis B Virus.

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >296 million people worldwide, including ∼850,000 in the USA, and kills 820,000 annually worldwide. Current nucleos(t)ide analogue (NA) or pegylated interferon α therapies do not eradicate the virus and would benefit from a complementary antiviral drug. We performed a preliminary screen of 28 dispirotripiperazines against HBV, identifying 9 hits with EC50 of 0.7-25 µM. Compound 11826096 displays the most potent activity and represents a promising lead for future optimization. While the mechanism of action is unknown, preliminary assays limit possible targets to activities involved in RNA accumulation, translation, capsid assembly, and/or capsid stability. In addition, we built machine learning models to determine if they were able to predict the activity of this series of compounds. The novelty of these molecules indicated they were outside of the applicability domain of these models.

Side Chain-Modified Benzothiazinone Derivatives with Anti-Mycobacterial Activity.

Tuberculosis (TB) is a leading infectious disease with serious antibiotic resistance. The benzothiazinone (BTZ) scaffold PBTZ169 kills Mycobacterium tuberculosis (Mtb) through the inhibition of the essential cell wall enzyme decaprenylphosphoryl-ß-D-ribose 2'-oxidase (DprE1). PBTZ169 shows anti-TB potential in animal models and pilot clinical tests. Although highly potent, the BTZ type DprE1 inhibitors in general show extremely low aqueous solubility, which adversely affects the drug-like properties. To improve the compounds physicochemical properties, we generated a series of BTZ analogues. Several optimized compounds had MIC values against Mtb lower than 0.01 µM. The representative compound 37 displays improved solubility and bioavailability compared to the lead compound. Additionally, compound 37 shows Mtb-killing ability in an acute infection mouse model.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

N-Phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine as a New Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitor.

Highly active antiretroviral therapy (HAART) has revolutionized human immunodeficiency virus (HIV) healthcare, turning it from a terminal to a potentially chronic disease, although some patients can develop severe comorbidities. These include neurological complications, such as HIV-associated neurocognitive disorders (HAND), which result in cognitive and/or motor function symptoms. We now describe the discovery, synthesis, and evaluation of a new class of N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) aimed at avoiding HAND. The most promising molecule, 12126065, exhibited antiviral activity against wild-type HIV-1 in TZM cells (EC50 = 0.24 nM) with low in vitro cytotoxicity (CC50 = 4.8 µM) as well as retained activity against clinically relevant HIV mutants. 12126065 also demonstrated no in vivo acute or subacute toxicity, good in vivo brain penetration, and minimal neurotoxicity in mouse neurons up to 10 µM, with a 50% toxicity concentration (TC50) of >100 µM, well below its EC50.

Antituberculosis Macozinone Extended-Release Tablets To Enhance Bioavailability: a Pilot Pharmacokinetic Study in Beagle Dogs.

Macozinone (MCZ; PBTZ169) is a first-in-class antituberculosis clinical-stage benzothiazinone-based drug candidate. Although its efficacy and safety have been strongly proven in several preclinical and clinical studies, the physicochemical and pharmacokinetic properties specific to MCZ required further optimization. Accordingly, this study aimed to evaluate the pharmacokinetics of MCZ administered as extended-release (ER) tablets F2 and F6 compared to immediate-release (IR) dispersible tablets for oral suspension. Oral absorption of MCZ from ER tablets was significantly different from that of IR tablets after a single oral dose in Beagle dogs in both fasted and fed states. In addition, food directly affects the bioavailability of MCZ from ER tablets but does not affect it from IR tablets. The high values of relative bioavailability of the prolonged-release tablets F2 and F6 compared to the IR tablets may indicate an indirect confirmation of their gastroretentive properties. Taken together, pharmacokinetic parameters have demonstrated that these MCZ oral formulations not just enhance drug bioavailability but may also improve regimen adherence by reducing MCZ dose frequency and reducing the development of drug resistance. IMPORTANCE Macozinone (MCZ) is the newest first-in-class clinical-stage benzothiazinone-based drug candidate for the treatment of tuberculosis. Yet, the extremely low oral bioavailability of MCZ, a major problem in clinical trials, needed to be addressed, and we are pleased to present our attempts to solve this issue. We report that extended-release tablets of MCZ significantly increased key pharmacokinetic parameters in the preclinical setting. We suggest that these MCZ oral formulations not just enhance drug bioavailability but may also improve regimen adherence by reducing MCZ dose frequency and reducing the development of drug resistance.

Multiple approaches to repurposing drugs for neuroblastoma.

Neuroblastoma (NB) is the second leading extracranial solid tumor of early childhood with about two-thirds of cases presenting before the age of 5, and accounts for roughly 15 percent of all pediatric cancer fatalities in the United States. Treatments against NB are lacking, resulting in a low survival rate in high-risk patients. A repurposing approach using already approved or clinical stage compounds can be used for diseases for which the patient population is small, and the commercial market limited. We have used Bayesian machine learning, in vitro cell assays, and combination analysis to identify molecules with potential use for NB. We demonstrated that pyronaridine (SH-SY5Y IC50 1.70 µM, SK-N-AS IC50 3.45 µM), BAY 11-7082 (SH-SY5Y IC50 0.85 µM, SK-N-AS IC50 1.23 µM), niclosamide (SH-SY5Y IC50 0.87 µM, SK-N-AS IC50 2.33 µM) and fingolimod (SH-SY5Y IC50 4.71 µM, SK-N-AS IC50 6.11 µM) showed cytotoxicity against NB. As several of the molecules are approved drugs in the US or elsewhere, they may be repurposed more readily for NB treatment. Pyronaridine was also tested in combinations in SH-SY5Y cells and demonstrated an antagonistic effect with either etoposide or crizotinib. Whereas when crizotinib and etoposide were combined with each other they had a synergistic effect in these cells. We have also described several analogs of pyronaridine to explore the structure-activity relationship against cell lines. We describe multiple molecules demonstrating cytotoxicity against NB and the further evaluation of these molecules and combinations using other NB cells lines and in vivo models will be important in the future to assess translational potential.

Thieno[2,3-d]pyrimidine-Core Compounds Show Activity against Clinically Relevant Gram-Positive Bacteria.

Thieno[2,3-d]pyrimidines represent a novel antibacterial prodrug scaffold, previously identified through a screening campaign against Mycobacterium tuberculosis in which the formation of highly antimycobacterial metabolites catalyzed by the nitroreductase Mrx2 is suggested to be the relevant killing mechanism. As analogous activation pathways may also be employed in other prokaryotes, in this work we explored general antibacterial effects of this compound class. Through exploration of the chemical space by different synthetic strategies, 51 novel derivatives were generated, biologically evaluated and thus enabled initial conclusions about structure-activity relationships. Remarkably, anti-Gram-positive activity can be well modulated, particularly towards Staphylococci (MRSA) and even slightly against some Gram-negative strains. The two most promising hit compounds showed good pharmacokinetic properties in vitro as well as acceptable toxicity in HeLa cells, qualifying them as starting points for lead-generation campaigns.

Pyronaridine Protects against SARS-CoV-2 Infection in Mouse.

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency-approved for use against severe acute respiratory coronavirus 2 (SARS-CoV-2). One of these is remdesivir, which was originally repurposed from its use against Ebola. We evaluated three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg and identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. The in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS-CoV-2-infected mice, reducing lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 µM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

A proof-of-concept study for the efficacy of dispirotripiperazine PDSTP in a rabbit model of herpes simplex epithelial keratitis.

Herpes simplex keratitis is an important infectious cause of blindness worldwide. The mainstay of antiviral therapy is treatment with long-established nucleoside analogues orally or topically. However, the emergence of resistant strains may become a major health concern in the future. Therefore, the development of backup antiherpetic medicines is urgently needed. Small molecule PDSTP is known to be active against herpes simplex type 1 strains in vitro, affecting early host-pathogen interactions. Here, we evaluated its preclinical efficacy in a rabbit model of herpes simplex epithelial keratitis. The mean course of keratitis and the corneal lesions in the 1.0% PDSTP gel group was statistically significantly less than in the negative control group and was comparable to that in the aciclovir group. These findings open up new opportunities for the development of antiherpetic drugs with an original mechanism of action.

Machine Learning Models for Mycobacterium tuberculosisIn Vitro Activity: Prediction and Target Visualization.

Tuberculosis (TB) is a major global health challenge, with approximately 1.4 million deaths per year. There is still a need to develop novel treatments for patients infected with Mycobacterium tuberculosis (Mtb). There have been many large-scale phenotypic screens that have led to the identification of thousands of new compounds. Yet, there is very limited investment in TB drug discovery which points to the need for new methods to increase the efficiency of drug discovery against Mtb. We have used machine learning approaches to learn from the public Mtb data, resulting in many data sets and models with robust enrichment and hit rates leading to the discovery of new active compounds. Recently, we have curated predominantly small-molecule Mtb data and developed new machine learning classification models with 18 886 molecules at different activity cutoffs. We now describe the further validation of these Bayesian models using a library of over 1000 molecules synthesized as part of EU-funded New Medicines for TB and More Medicines for TB programs. We highlight molecular features which are enriched in these active compounds. In addition, we provide new regression and classification models that can be used for scoring compound libraries or used to design new molecules. We have also visualized these molecules in the context of known molecular targets and identified clusters in chemical property space, which may aid in future target identification efforts. Finally, we are also making these data sets publicly available, representing a significant increase to the available Mtb inhibition data in the public domain.

Development of Machine Learning Models and the Discovery of a New Antiviral Compound against Yellow Fever Virus.

Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by infected mosquitoes. Large epidemics of YF occur when the virus is introduced into heavily populated areas with high mosquito density and low vaccination coverage. The lack of a specific small molecule drug treatment against YF as well as for homologous infections, such as zika and dengue, highlights the importance of these flaviviruses as a public health concern. With the advancement in computer hardware and bioactivity data availability, new tools based on machine learning methods have been introduced into drug discovery, as a means to utilize the growing high throughput screening (HTS) data generated to reduce costs and increase the speed of drug development. The use of predictive machine learning models using previously published data from HTS campaigns or data available in public databases, can enable the selection of compounds with desirable bioactivity and absorption, distribution, metabolism, and excretion profiles. In this study, we have collated cell-based assay data for yellow fever virus from the literature and public databases. The data were used to build predictive models with several machine learning methods that could prioritize compounds for in vitro testing. Five molecules were prioritized and tested in vitro from which we have identified a new pyrazolesulfonamide derivative with EC50 3.2 µM and CC50 24 µM, which represents a new scaffold suitable for hit-to-lead optimization that can expand the available drug discovery candidates for YF.

Discovery of 5-Nitro-6-thiocyanatopyrimidines as Inhibitors of Cryptococcus neoformans and Cryptococcus gattii.

Opportunistic infections from pathogenic fungi present a major challenge to healthcare because of a very limited arsenal of antifungal drugs, an increasing population of immunosuppressed patients, and increased prevalence of resistant clinical strains due to overuse of the few available antifungals. Cryptococcal meningitis is a life-threatening opportunistic fungal infection caused by one of two species in the Cryptococcus genus, Cryptococcus neoformans and Cryptococcus gattii. Eighty percent of cryptococcosis diseases are caused by C. neoformans that is endemic in the environment. The standard of care is limited to old antifungals, and under a high standard of care, mortality remains between 10 and 30%. We have identified a series of 5-nitro-6-thiocyanatopyrimidine antifungal drug candidates using in vitro and computational machine learning approaches. These compounds can inhibit C. neoformans growth at submicromolar levels, are effective against fluconazole-resistant C. neoformans and a clinical strain of C. gattii, and are not antagonistic with currently approved antifungals.

6,11-Dioxobenzo[f]pyrido[1,2-a]indoles Kill Mycobacterium tuberculosis by Targeting Iron-Sulfur Protein Rv0338c (IspQ), A Putative Redox Sensor.

Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum. On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPI-resistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centers and putative iron-sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe-2S domain in IspQ and another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit-target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.

The past, present and future of RNA respiratory viruses: influenza and coronaviruses.

Influenza virus and coronaviruses continue to cause pandemics across the globe. We now have a greater understanding of their functions. Unfortunately, the number of drugs in our armory to defend us against them is inadequate. This may require us to think about what mechanisms to address. Here, we review the biological properties of these viruses, their genetic evolution and antiviral therapies that can be used or have been attempted. We will describe several classes of drugs such as serine protease inhibitors, heparin, heparan sulfate receptor inhibitors, chelating agents, immunomodulators and many others. We also briefly describe some of the drug repurposing efforts that have taken place in an effort to rapidly identify molecules to treat patients with COVID-19. While we put a heavy emphasis on the past and present efforts, we also provide some thoughts about what we need to do to prepare for respiratory viral threats in the future.