Small molecules restore azole activity against drug-tolerant and drug-resistant Candida isolates.

Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.

Acyldepsipeptide Antibiotics and a Bioactive Fragment Thereof Differentially Perturb Mycobacterium tuberculosis ClpXP1P2 Activity in Vitro.

Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.

Hydroxyl-Rich Hydrophilic Endocytosis-Promoting Peptide with No Positive Charge.

Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.

Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections.

Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti-Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity profiles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efficacy against Leishmania infections. Structure-activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efficacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo(ethylene glycol) methacrylate-co-pentafluorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efficacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efficacy of anti-Leishmania compounds.

Discovery of Retro-1 Analogs Exhibiting Enhanced Anti-vaccinia Virus Activity.

Orthopoxviruses (OPXVs) are an increasing threat to human health due to the growing population of OPXV-naive individuals after the discontinuation of routine smallpox vaccination. Antiviral drugs that are effective as postexposure treatments against variola virus (the causative agent of smallpox) or other OPXVs are critical in the event of an OPXV outbreak or exposure. The only US Food and Drug Administration-approved drug to treat smallpox, Tecovirimat (ST-246), exerts its antiviral effect by inhibiting extracellular virus (EV) formation, thereby preventing cell-cell and long-distance spread. We and others have previously demonstrated that host Golgi-associated retrograde proteins play an important role in monkeypox virus (MPXV) and vaccinia virus (VACV) EV formation. Inhibition of the retrograde pathway by small molecules such as Retro-2 has been shown to decrease VACV infection in vitro and to a lesser extent in vivo. To identify more potent inhibitors of the retrograde pathway, we screened a large panel of compounds containing a benzodiazepine scaffold like that of Retro-1, against VACV infection. We found that a subset of these compounds displayed better anti-VACV activity, causing a reduction in EV particle formation and viral spread compared to Retro-1. PA104 emerged as the most potent analog, inhibiting 90% viral spread at 1.3 µM with a high selectivity index. In addition, PA104 strongly inhibited two distinct ST-246-resistant viruses, demonstrating its potential benefit for use in combination therapy with ST-246. These data and further characterizations of the specific protein targets and in vivo efficacy of PA104 may have important implications for the design of effective antivirals against OPXV.

Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins.

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

Analysis of vertebrate vision in a 384-well imaging system.

Visual impairment affects 253 million people worldwide and new approaches for prevention and treatment are urgently needed. While small molecules with potential beneficial effects can be examined in various model systems, the in vivo evaluation of visual function remains a challenge. The current study introduces a novel imaging system for measuring visually-guided behaviors in larval zebrafish. The imaging system is the first to image four 96-well plates with a single camera for automated measurements of activity in a 384-well format. In addition, it is the first system to project moving visual stimuli and analyze the optomotor response in the wells of a 96-well plate. We found that activity is affected by tricaine, diazepam and flumazenil. Surprisingly, diazepam treatments induce a loss of visual responses, at concentrations that do not affect activity or induce hyperactivity. Overall, our studies show that the developed imaging system is suitable for automated measurements of vertebrate vision in a high-throughput format.

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish.

A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor.

In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the ß-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 µM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.

Structurally optimized analogs of the retrograde trafficking inhibitor Retro-2cycl limit Leishmania infections.

In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58µM and10.57+/-2.66µM, respectively, which is significantly lower than 40.15µM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.

Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome.

The 20S core particle of the proteasome in Mycobacterium tuberculosis (Mtb) is a promising, yet unconventional, drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. Selective inhibitors of the Mtb 20S were designed and synthesized on the bases of both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogues weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.

A Conformationally Constrained Cyclic Acyldepsipeptide Is Highly Effective in Mice Infected with Methicillin-Susceptible and -Resistant Staphylococcus aureus.

BACKGROUND: Cyclic acyldepsipeptides (ADEPs) are a novel class of antibacterial agents, some of which (e.g., ADEP 4) are highly active against Gram-positive bacteria. The focus of these in vivo studies is ADEP B315, a rationally designed compound that has the most potent in vitro activity of any ADEP analog reported to date. METHODS: In vivo efficacy experiments were performed using lethal intraperitoneal mice infection models with a methicillin-sensitive S. aureus (MSSA) and a methicillin-resistant (MRSA) strain. The infected mice were treated with ADEP B315, a des-methyl analog of ADEP 4, vancomycin, or the vehicle used for the ADEPs and their survival was assessed daily. A subset of MSSA-infected mice was sacrificed soon after inoculation and the bacterial burden was measured in their livers and spleens. The toxicity of ADEP B315 was assessed in viability assays using human whole blood cultures. RESULTS: In the MSSA experiments, all mice treated with the vehicle succumbed to the infection within 24 hours. All tested compounds were effective in prolonging survival of infected mice (p<0.001). Mice treated with ADEP B315 had a 39% survival rate by 10 days compared to 7% survival in mice treated with a des-methyl ADEP 4 analog (p = 0.017). Survival of the infected mice treated with ADEP B315 was comparable to those treated with vanocmycin (p = 0.12) at the same dose. Further, bacterial burden in the liver and spleen was significantly lower in mice treated with ADEP B315 compared to controls. In the MRSA experiments, ADEP B315 was able to significantly prolong survival compared to mice treated with either the vehicle (p = 0.001) or vancomycin (p = 0.007). ADEP B315 exhibited no significant toxicity in human whole blood cultures at concentrations up to 25 µg/ml. CONCLUSIONS: ADEP B315 is safe and can cure mice that have lethal infections of methicillin-sensitive and -resistant strains of S. aureus.

Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle.

The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.

Substrate-guided optimization of the syringolins yields potent proteasome inhibitors with activity against leukemia cell lines.

Natural products that inhibit the proteasome have been fruitful starting points for the development of drug candidates. Those of the syringolin family have been underexploited in this context. Using the published model for substrate mimicry by the syringolins and knowledge about the substrate preferences of the proteolytic subunits of the human proteasome, we have designed, synthesized, and evaluated syringolin analogs. As some of our analogs inhibit the activity of the proteasome with second-order rate constants 5-fold greater than that of the methyl ester of syringolin B, we conclude that the substrate mimicry model for the syringolins is valid. The improvements in in vitro potency and the activities of particular analogs against leukemia cell lines are strong bases for further development of the syringolins as anti-cancer drugs.

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase.

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N-acylphenylalanine is thought to structurally mimic the ClpP-binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N-acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N-capped with methyl-branched acyl groups-like the side chains of residues in the (I/L)GF motifs-were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N-acylphenylalanine moiety is not simply a stand-in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl-branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.

Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor.

UNLABELLED: Pupylation is a posttranslational modification peculiar to actinobacteria wherein proteins are covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated protein degradation in mycobacteria. Here, we report studies of pupylation in a streptomycete that is phylogentically related to mycobacteria. We constructed mutants of Streptomyces coelicolor lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these mutants share a high susceptibility to oxidative stress compared to that of the wild-type strain. Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking genes encoding the 20S proteasome. Our findings shed new light on this unusual posttranslational modification and its role in Streptomyces physiology. IMPORTANCE: The presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have motivated reevaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome system has been studied extensively in mycobacteria, it is much less understood in streptomycetes, members of a large genus of actinobacteria known for highly choreographed life cycles in which phases of morphological differentiation, sporulation, and secondary metabolism are often regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces coelicolor for the first time and present new evidence that links pupylation and the oxidative stress response in this bacterium. Surprisingly, we found that the Pup ligase has a Pup-independent role in sporulation.

Fragment-Based Strategy for Investigating and Suppressing the Efflux of Bioactive Small Molecules.

Membrane protein-mediated drug efflux is a phenomenon that compromises our ability to treat both infectious diseases and cancer. Accordingly, there is much interest in the development of strategies for suppression of the mechanisms by which therapeutic agents are effluxed. Here, using resistance to the cyclic acyldepsipeptide (ADEP) antibacterial agents as a model, we demonstrate a new counter-efflux strategy wherein a fragment of an actively exported bioactive compound competitively interferes with its efflux and potentiates its activity. A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimized ADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor. Beyond their validation of a new approach to studying molecular recognition by drug efflux pumps, our findings have important implications for killing Mycobacterium tuberculosis with ADEPs and reclaiming the efficacies of therapeutic agents whose activity has been compromised by efflux pumps.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery.

Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.

A simple fragment of cyclic acyldepsipeptides is necessary and sufficient for ClpP activation and antibacterial activity.

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.

Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses.

Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2(cycl), an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2(cycl) and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure-activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry.