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Proteasomes are compartmentalized, ATP-dependent, N-terminal nucleophile hydrolases that play essentials roles in intracellular protein turnover. They are present in all 3 kingdoms. Pharmacological inhibition of proteasomes is detrimental to cell viability. Proteasome inhibitor rugs revolutionize the treatment of multiple myeloma. Proteasomes in pathogenic microbes such as Mycobacterium tuberculosis (Mtb), Plasmodium falciparum (Pf), and other parasites and worms have been validated as therapeutic targets. Starting with Mtb proteasome, efforts in developing inhibitors selective for microbial proteasomes have made great progress lately. In this review, we describe the strategies and pharmacophores that have been used in developing proteasome inhibitors with potency and selectivity that spare human proteasomes and highlight the development of clinical proteasome inhibitor candidates for treatment of leishmaniasis and Chagas disease. Finally, we discuss the future challenges and therapeutical potentials of the microbial proteasome inhibitors.

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum., Author summary The Apicomplexa phylum comprises thousands of parasitic species, among which the causative agents of malaria, toxoplasmosis and cryptosporidiosis. New parasiticidal compounds and drugs are urgently needed for treatment of these devastating diseases. As these parasites have adapted innovative pathways for nutrient acquisition, several studies have investigated vitamins and cofactor synthesis and salvage, with the aim of identifying unexplored drug targets. Coenzyme A (CoA) is an essential cofactor for cell biology and is synthesized from pantothenate (Pan, vitamin B5). The discovery of the druggability of CoA synthesis in Plasmodium falciparum has sparked intensive research toward lead compounds identification and preclinical development. Here, we review the current literature on the topic from biological and pharmacological perspectives. Focusing on Plasmodium species and Toxoplasma gondii, we describe recent findings on the importance of Pan synthesis, salvage, and metabolization to CoA in this phylum. In addition, we summarize recent promising advances in the exploration and exploitation of these pathways for lead compounds optimization and drug development.

Infection with avian influenza A H5N1 virus results in acute lung injury (ALI) and has a high mortality rate (52.79%) because there are limited therapies available for treatment. Drug repositioning is an economical approach to drug discovery. We developed a method for drug repositioning based on high-throughput RNA sequencing and identified several drugs as potential treatments for avian influenza A H5N1 virus. Using high-throughput RNA sequencing, we identified a total of 1,233 genes differentially expressed in A549 cells upon H5N1 virus infection. Among these candidate genes, 79 drug targets (corresponding to 59 approved drugs) overlapped with the DrugBank target database. Twenty-two of the 41 commercially available small-molecule drugs reduced H5N1-mediated cell death in cultured A549 cells, and fifteen drugs that protected A549 cells when administered both pre- and post-infection were tested in an H5N1-infection mouse model. The results showed significant alleviation of acute lung injury by amitriptyline HCl (an antidepressant drug), flavin adenine dinucleotide (FAD; an ophthalmic agent for vitamin B2 deficiency), azacitidine (an anti-neoplastic drug) and calcitriol (an active form of vitamin D). All four agents significantly reduced the infiltrating cell count and decreased the lung injury score in H5N1 virus-infected mice based on lung histopathology, significantly improved mouse lung edema by reducing the wet-to-dry weight ratio of lung tissue and significantly improved the survival of H5N1 virus-infected mice. This study not only identifies novel potential therapies for influenza H5N1 virus-induced lung injury but also provides a highly effective and economical screening method for repurposing drugs that may be generalizable for the prevention and therapy of other diseases., Author summary Highly pathogenic avian influenza (HPAI) A virus H5N1 causes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), with mortality as high as 52.79%. No vaccine for HPAI virus is available, and current treatments for influenza A H5N1 virus-induced ALI have limitations. Drug repurposing may be an effective approach for developing novel therapeutic strategies. In this study, we identified 4 drugs, the antidepressant amitriptyline HCl, the ophthalmic flavin adenine dinucleotide, the anti-neoplastic azacitidine and the vitamin D-deficiency treatment calcitriol, as being highly effective for the treatment of H5N1 virus-induced ALI using a transcriptomic-based high-throughput repurposing drug screening. These approved drugs might constitute novel potential remedies for treating influenza H5N1 virus infection, and this screening method may be generalizable for drug repositioning to identify new indications for other diseases.

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions., Author summary The underlying cause of disease in a group of rapidly progressing neurodegenerative disorders called prion diseases is the misfolding of the prion protein (PrP) into a conformation that can self-template and spread disease throughout the brain. Diseases caused by this phenomenon include Creutzfeldt–Jakob disease (CJD), chronic wasting disease, and bovine spongiform encephalopathy (“mad cow” disease). In 2015, we demonstrated that this same mechanism is responsible for the neurodegenerative disease multiple system atrophy (MSA); however, the disease is caused by the misfolding of the protein α-synuclein rather than PrP. Having shown that α-synuclein prions in MSA patient samples exhibit a number of properties consistent with PrP prions in CJD patients, we sought to establish and define a rigorous transgenic mouse model of α-synuclein prion propagation to support ongoing drug discovery efforts for MSA therapeutics. In this study, we identified optimized methods for transmitting MSA in a transgenic mouse model of α-synuclein prion spreading and defined disease pathogenesis in these mice. These results are needed to properly evaluate compounds that may prevent α-synuclein prion spreading. We also showed that in both human and mouse brain, α-synuclein prion spreading precedes the formation of neuropathological lesions traditionally used to define disease, yielding new insights into the progression of MSA.

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization’s Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB., Author summary More humans die of tuberculosis (TB) than any other infectious disease, yet diagnostic tools remain limited. Here, we used the Diversity Outbred mouse population to discover candidate protein biomarkers of human TB. By applying statistical methods and machine learning to multidimensional data, we identified CXCL1 and MMP8 as the two most promising protein biomarker candidates. When evaluated in samples from human patients, CXCL1 achieved the World Health Organization’s targeted profile for a triage diagnostic test, discriminating active TB from important clinical differential diagnoses: latent Mtb infection and non-TB lung disease in HIV-negative adults. Overall, our studies show how a translationally relevant animal population model can accelerate TB biomarker discovery, validation, and testing for humans.

It is estimated that more than 1 billion people across the world are affected by a neglected tropical disease (NTD) that requires medical intervention. These diseases tend to afflict people in areas with high rates of poverty and cost economies billions of dollars every year. Collaborative drug discovery efforts are required to reduce the burden of these diseases in endemic regions. The release of “Open Access Boxes” is an initiative launched by Medicines for Malaria Venture (MMV) in collaboration with its partners to catalyze new drug discovery in neglected diseases. These boxes are mainly requested by biology researchers across the globe who may not otherwise have access to compounds to screen nor knowledge of the workflow that needs to be followed after identification of actives from their screening campaigns. Here, we present guidelines on how to move such actives beyond the hit identification stage, to help in capacity strengthening and enable a greater impact of the initiative.

Human parasitic nematodes are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), diseases that are endemic to more than 80 countries and that consistently rank in the top ten for the highest number of years lived with disability. These filarial nematodes have evolved an obligate mutualistic association with an intracellular bacterium, Wolbachia, a symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Elimination of the bacteria causes adult worms to die, making Wolbachia a primary target for developing new interventional tools to combat filariases. To further explore Wolbachia as a promising indirect macrofilaricidal drug target, the essential cellular processes that define the symbiotic Wolbachia-host interactions need to be identified. Genomic analyses revealed that while filarial nematodes encode all the enzymes necessary for glycolysis, Wolbachia does not encode the genes for three glycolytic enzymes: hexokinase, 6-phosphofructokinase, and pyruvate kinase. These enzymes are necessary for converting glucose into pyruvate. Wolbachia, however, has the full complement of genes required for gluconeogenesis starting with pyruvate, and for energy metabolism via the tricarboxylic acid cycle. Therefore, we hypothesized that Wolbachia might depend on host glycolysis to maintain a mutualistic association with their parasitic host. We did conditional experiments in vitro that confirmed that glycolysis and its end-product, pyruvate, sustain this symbiotic relationship. Analysis of alternative sources of pyruvate within the worm indicated that the filarial lactate dehydrogenase could also regulate the local intracellular concentration of pyruvate in proximity to Wolbachia and thus help control bacterial growth via molecular interactions with the bacteria. Lastly, we have shown that the parasite’s pyruvate kinase, the enzyme that performs the last step in glycolysis, could be a potential novel anti-filarial drug target. Establishing that glycolysis is an essential component of symbiosis in filarial worms could have a broader impact on research focused on other intracellular bacteria-host interactions where the role of glycolysis in supporting intracellular survival of bacteria has been reported., Author summary Wuchereria bancrofti, Brugia malayi, Brugia timori, and Onchocerca volvulus are human parasitic nematodes and causative agents of lymphatic filariasis (LF, elephantiasis) and onchocerciasis (river blindness). These neglected tropical diseases are ranked in the top ten for the highest years lived with disability. In 2016, the global prevalence of LF was between 24.8 to 36.1 million while for onchocerciasis it was 9.5 to 24.2 million. These nematodes have evolved a mutualistic association with an intracellular bacterium, Wolbachia, an obligatory symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Consequently, these bacteria are a target of interest for developing new interventional tools to combat the filariases. To develop macrofilaricidal drug targets, we must identify the cellular processes that define the symbiotic relationship between the bacteria and their filarial hosts. Here, we evaluated the role of glycolysis—a pathway that is incomplete in Wolbachia—in the symbiotic relationship. We found that glycolysis is an essential metabolic pathway that supports the symbiosis between the filarial worm and Wolbachia, and demonstrated that this pathway, on which Wolbachia rely, can be exploited to discover new or repurposed drugs, and thus facilitate anti-filarial elimination goals.

Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl-) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K+) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K+ levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K+, Cl-, and pH. We demonstrate that intraphagosomal K+ levels increase during macrophage phagosome maturation, and find using a novel fluorescent K+-responsive reporter Mtb strain that K+ is not limiting during macrophage infection. Disruption of Mtb K+ homeostasis by deletion of the Trk K+ uptake system results in dampening of the bacterial response to pH and Cl-, and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb., Author summary Tuberculosis is the leading cause of death from infectious diseases globally, and knowledge of the fundamental biology underlying successful host colonization by Mycobacterium tuberculosis (Mtb) is critical for understanding its pathogenicity. Our study focuses on one such important facet—the ability of Mtb to sense and respond to changing environmental ionic signals, and its relation to maintenance of bacterial ionic homeostasis. Potassium (K+) is the most abundant ion in both mammalian and bacterial cells, but much remains unknown about how it may affect host-pathogen interactions. We show here that Mtb has distinct gene expression changes in response to variation in environmental K+ levels. Importantly, disruption of a Mtb K+ uptake system reduces the ability of Mtb to respond to other key ionic cues, and results in diminished host colonization. Our study reveals novel roles of K+ during Mtb-host interactions, and suggests the broader importance of abundant ions in bacterial pathogenicity.

Efforts at host-directed therapy of tuberculosis have produced little control of the disease in experimental animals to date. This is not surprising, given that few specific host targets have been validated, and reciprocally, many of the compounds tested potentially impact multiple targets with both beneficial and detrimental consequences. This puts a premium on identifying appropriate molecular targets and subjecting them to more selective modulation. We discovered an aminopyrimidine small molecule, 2062, that had no direct antimycobacterial activity, but synergized with rifampin to reduce bacterial burden in Mtb infected macrophages and mice and also dampened lung immunopathology. We used 2062 and its inactive congeners as tool compounds to identify host targets. By biochemical, pharmacologic, transcriptomic and genetic approaches, we found that 2062’s beneficial effects on Mtb control and clearance in macrophages and in mice are associated with activation of transcription factor EB via an organellar stress response. 2062-dependent TFEB activation led to improved autophagy, lysosomal acidification and lysosomal degradation, promoting bacterial clearance in macrophages. Deletion of TFEB resulted in the loss of IFNγ-dependent control of Mtb replication in macrophages. 2062 also targeted multiple kinases, such as PIKfyve, VPS34, JAKs and Tyk2, whose inhibition likely limited 2062’s efficacy in vivo. These findings support a search for selective activators of TFEB for HDT of TB., Author summary Tuberculosis (TB) is now the leading cause of death from a single infection. Effective TB treatments take months, are toxic and lead to the development of multidrug- (MDR) and extensively drug-resistant (XDR) TB. Host-directed therapies (HDT) are being explored for their potential to enhance the efficacy of anti-mycobacterial agents and reduce lung pathology. Rifampin is one of the most important TB drugs but many patients taking the standard dose have suboptimal plasma levels. We discovered an aminopyrimidine compound with no direct antimycobacterial activity that enhanced control of Mycobacterium tuberculosis (Mtb) in combination with low-dose rifampin in macrophages and in mice. Beneficial effects correlated with the activation of the host transcription factor EB, a master regulator of lysosomal biogenesis and lysosomal activation. Selective activators of host TFEB may aid in the management of TB in patients with suboptimal plasma levels of rifampin.

Stewart Cole and colleagues determined the complete genome sequence of Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), in 1998 [1]. This was a landmark achievement that heralded a new age in TB drug discovery. With the genome sequence in hand, drug discoverers suddenly had thousands of new potential targets to explore. But the excitement has since faded [2]. It is unquestioned that genomics has transformed our understanding of the biology of this pathogen. However, the expectation that the Mtb genome sequence would rapidly lead to new therapeutic interventions remains unfulfilled [3]. One of the (many) reasons for this unrealized potential is that our tools to systematically interrogate the Mtb genome and its drug targets—so-called functional genomics—have been limited. In this Pearl, I argue that the recent development of robust CRISPR-based genetics in Mtb [4] overcomes many prior limitations and holds the potential to close the gap between genomics and TB drug discovery.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

As a molecular biologist, you start by learning the basics of the DNA code. Reading and translating the code come first, and then you can move on to doing your own careful editing using cut and paste and copy and paste. Once you’ve acquired these skills, you can manipulate genes, genomes, and proteins to examine how cells “work” and to adjust the way they work. The power, versatility, and affordability of these techniques have improved in leaps and bounds in recent years, in parallel with improvements in information technologies. For example, DNA sequencing, or the ability to “read” an entire genome, is now accessible to any reasonably well-resourced research team. The techniques are important, but it’s really what you do with them that can capture the imagination. The discovery of key disease-associated genes, often many years after the consequences of their action, or inaction, are known, has been a source of inspiration to me personally. In particular, I recall reading about identification of the cystic fibrosis gene in 1989, when starting my PhD, and hearing about identification of the leptin gene, linked to obesity, as a post-doc in 1995. Then, I saw what impact gene discovery could have on my own molecular parasitology community: the malaria parasite var genes in 1995, the African trypanosome human serum resistance-associated gene in 1998, and the malaria parasite chloroquine resistance transporter in 2000 are just a few examples. It remains challenging, though, to identify genes involved in a particular process of interest. Discovery of a new and important gene is also only the first step in research aimed at fully understanding function and potential as a therapeutic target or a vaccination candidate, for example. The outstanding challenges, however, are exactly what continue to drive technological innovation in the life sciences. So what’s the situation for the unicellular trypanosomatid parasites we work on in Dundee? These highly motile cells, about 1/50th of a millimetre long, are spread between mammals by blood-sucking insects. They have a devastating impact on the world’s poor, causing the “neglected tropical diseases” African trypanosomiasis, Chagas disease, and the leishmaniases. The consequences of this range of human and animal diseases are hundreds of thousands of deaths each year and around a million cases a year of the disfiguring lesions associated with cutaneous leishmaniasis. Trypanosomes also severely curtail the import of susceptible horses, cattle, sheep, and goats across Africa. We’ve focussed on African trypanosomes in particular and see that antiparasitic adaptations in the host and antiparasitic drugs are constantly threatened by obsolescence in the “arms race” between us and the parasites. In the hope of helping to gain the upper hand in this race, we adapted a genetic screening approach that allows us to survey every parasite gene for roles in drug action and drug resistance. This revealed >50 new genes linked to drugs in clinical use. The drugs nifurtimox and eflornithine, for example, are widely used as a combination therapy. We confirmed that nifurtimox is converted to a more potent drug by a parasite enzyme and, as also reported independently by other groups, found that eflornithine is taken up by a parasite amino acid transporter. A much older drug, suramin, was found to hitch-a-ride into the cell on a parasite surface protein. This was a particularly satisfying discovery because Paul Ehrlich, “the father of chemotherapy,” who developed both the “magic bullet” hypothesis and the precursors to suramin over 100 years ago, stated, “When once we are acquainted with the majority of the chemioreceptors of a particular kind of parasite…we shall have far-reaching possibilities of simultaneous attack by various agencies” (Ehrlich, 1913). So, we found one of Ehrlich’s “chemioreceptors,” and this does indeed present new opportunities for therapeutic development. Another finding related to two more old drugs for which cross-resistance had been reported over 60 years ago. This melarsoprol and pentamidine cross-resistance was linked to a defect in an aquaglyceroporin, another parasite membrane transporter. Resistance to melarsoprol, a rather toxic but otherwise effective arsenic-containing drug, had been observed in up to 50% of patients in some areas. Teams that collected and stored resistant parasites from across Africa were able to rapidly identify defects in this transporter and to trace them back to the mid-1970s. Thus, the genetic changes that allowed trypanosomes to resist this particular therapy are now known, the impact of these changes can be investigated in detail, and the spread and distribution of resistant parasites can be effectively monitored. It’s quite remarkable how biological processes frequently become experimentally accessible in unanticipated ways, probably because we still have so much to learn about many of them. This has also been my personal experience. In the case of our own findings, it was our work on DNA repair that allowed us to develop the genetic screening approach described above. In addition, our initial motivation for developing this approach was a desire to tackle a completely different gene expression question; fortunately, we were able to initiate collaborations with colleagues who were already established experts in drug resistance. What is clear is that knowing how drugs work and how they stop working helps improve the prospects of devising more effective and durable therapies and also improves prospects for monitoring and tackling resistance when it arises. As primarily “basic researchers,” working with partners in the Dundee Drug Discovery Unit, we will continue our efforts to discover and dissect druggable biology in the trypanosomatids. Indeed, tools and technologies are now in place to probe these chemical–biology interactions at higher throughput than ever before. Since our work continuously relies upon the work and discoveries of many colleagues—too many to mention, I’m afraid—I end by reiterating what’s probably rather obvious: impact against these and other human and animal pathogens requires a community of researchers, critical mass, open interaction, collaboration, and access to powerful technology, at both basic and translational levels. What is also clear is that experiments with pathogens will continue to yield insights of far broader relevance to the life sciences and beyond. Image 1 David Horn.

Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS) virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS) and structure-activity relationship (SAR) analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR); inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections., Author Summary Antibiotic resistant and tolerant bacterial pathogens are responsible for acute, chronic and persistent human infections recalcitrant to any current treatments. Therefore, there is an urgent need to identify new antimicrobial drugs that will help circumvent the current antibiotic resistance crisis. Bacterial pathogens often develop resistance to antibiotic drugs that target bacterial growth or viability. In contrast, strategies that specifically target virulence pathways non-essential for growth could limit selective resistance, and thus are candidates for the development of next-generation antimicrobial therapeutics. In this study we target the bacterial communication system MvfR (PqsR), which is known to control virulence of the opportunistic bacterial pathogen Pseudomonas aeruginosa. We identified and improved upon new small molecules that effectively silence the MvfR communication system, and as a result block P. aeruginosa virulence both in vitro and in vivo. Moreover, these new compounds are the first known to restrict the ability of bacteria to form antibiotic-tolerant cells and consequently proved to be very effective at preventing persistent infection in a mammalian infection model. Because of their ability to simultaneously block acute and persistent infections, these new molecules may provide a very strong basis for the development of next generation antimicrobials.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection., Author Summary Alphaviruses occur worldwide, causing significant diseases such as encephalitis or arthritis in humans and animals. In addition, some alphaviruses, such as VEEV, pose a biothreat due to their high infectivity and lack of available treatments. To discover small molecule inhibitors with lead development potential, we used a cell-based assay to screen 348,140 compounds for inhibition of a VEEV-induced cytopathic effect. The screen revealed a scaffold with high inhibitory VEEV cellular potency and low cytotoxicity liability. While most previously reported anti-alphavirus compounds inhibit host proteins, evidence supported that this scaffold targeted the VEEV nsP2 protein, and that inhibition was associated with viral replication. Interestingly, compound resistance studies with VEEV mapped activity to the N-terminal domain of nsP2, to which no known function has been attributed. Ultimately, this discovery has delivered a small molecule-derived class of potent VEEV inhibitors whose activity is coupled to the nsP2 viral protein, a novel target with a previously unestablished biological role that is now implicated in viral replication.

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4+ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation)., Author Summary To contain the HIV-1 epidemic, it is necessary to develop antivirals that prevent HIV-1 transmission. It is well known that HIV infection might be accompanied by other pathogens, which often are engaged with HIV-1 in a vicious circle of mutual facilitation. One of the most common of these pathogens is herpes simplex virus (HSV) type 2. Since there is an urgent need for a next generation antivirals that are multi-targeted, we can now report on the development of the first antiviral of this new generation that efficiently suppresses both HIV-1 and HSV-2. We found that the dual-targeted antiviral drug affects several targets for viral replication. It uniquely combines in one molecule three important abilities: (i) to efficiently suppress HSV-encoded DNA polymerase, (ii) to efficiently suppress HIV-1-encoded reverse transcriptase, and (iii) to stimulate secretion of CC-chemokines that downregulate the HIV-1 coreceptor CCR5. The compound suppresses both viruses in a wide-range of in vitro, ex vivo, and in vivo experimental models. The ability of one molecule to suppress HIV-1 and HSV-2 by combining direct activity against their two key enzymes and indirect immunomodulatory effects is unique in the antiviral field.

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development., Author Summary Current therapy for HBV blocks DNA synthesis by the viral reverse transcriptase and can control the infection indefinitely, but treatment rarely cures patients. More patients could be cured by suppressing HBV replication further using a new drug in combination with the existing ones. The HBV RNAseH is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. We expressed active recombinant HBV RNAseHs and demonstrated that it was suitable for antiviral drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified based on antagonists of the HIV RNAseH and integrase enzymes. Twelve of these compounds inhibited the HBV RNAseH in enzymatic assays, and one inhibited HBV replication in cell-based assays. The high percentage of compounds developed against the HIV RNAseH and integrase that were also active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can be used to guide anti-HBV RNAseH drug discovery.

Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors., Author Summary Like other viruses, HIV has a limited genome and needs to exploit the machinery of the host cell to complete its replication cycle. The elucidation of virus-host interactions not only sheds light on pathogenesis but also provides opportunities in a limited number of cases to develop novel antiviral drugs. A prototypical example is the interaction between the cellular protein LEDGF/p75 and HIV-1 integrase (IN). Here we generated a human somatic LEDGF/p75 knockout cell line to demonstrate that HIV-1 replication is highly dependent on its cofactor. We show that the residual replication of laboratory strains is predominantly mediated by a LEDGF/p75-related protein, HRP-2. Interestingly, the recently developed HIV-1 IN inhibitors that target the LEDGF/p75-IN interaction interface, LEDGINs, remain active even in the absence of LEDGF/p75. We demonstrate that LEDGINs efficiently block the interaction between IN and HRP-2. In case HIV-1 would be able to bypass LEDGF/p75-dependent replication using HRP-2 as an alternative tether, LEDGINs would remain fully active.

HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1) – or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care., Author Summary Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver cancer and liver transplantation worldwide. No vaccine is available for preventing the spread of HCV, and the current therapeutic regimen is only moderately effective and causes serious side effects. New antiviral agents are required to treat HCV infection, but the high mutation rate of HCV hinders the effectiveness of virus-specific inhibitors. Targeting the host enzymes required for HCV to replicate offers a promising new direction for antiviral therapy. During infection, HCV promotes excessive fat accumulation in the liver, which benefits the virus as this promotes formation of lipid droplets, a cellular organelle essential for assembly of new HCV infectious viral particles. Here, we report the development of a specific inhibitor targeting SKI-1/S1P, a host enzyme required for lipid production in human cells. We show that inhibiting SKI-1/S1P activity in human liver cells effectively blocks lipid droplet formation and HCV infection. Many prevalent human viruses, such as dengue, rotavirus, and hepatitis B virus, hijack host lipid metabolic pathways similar to those targeted by HCV to complete their lifecycle. Thus, we propose that cellular SKI-1/S1P is a potential target for developing desperately needed novel broad-spectrum antiviral drugs.