Challenges Based on Antiplasmodial and Antiviral Activities of 7-Chloro-4-aminoquinoline Derivatives.

We report the structural functionalization of the terminal amino group of N1 -(7-chloroquinolin-4-yl) butane-1,4-diamine, leading to a series of 7-chloro-4-aminoquinoline derivatives, and their evaluation as potent anti-malarial and anti-viral agents. Some compounds exhibited promising anti-malarial effects against the Plasmodium falciparum 3D7 (chloroquine-sensitive) and Dd2 (chloroquine-resistant) strains. In addition, these compounds were assayed in vitro against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compound 5 h, bearing an N-mesityl thiourea group, displayed pronounced anti-infectious effects against malaria, IAV, and SARS-CoV-2. These results provide new insights into drug discovery for the prevention or treatment of malaria and virus co-infection.

Conformation-Dependent Influences of Hydrophobic Amino Acids in Two In-Register Parallel ß-Sheet Amyloids, an α-Synuclein Amyloid and a Local Structural Model of PrPSc.

Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc underlies the existence of many prion strains and species barriers of prions, although the conformational information is extremely limited. Interestingly, differences between polymorphic or species-specific residues responsible for the species/strain barriers are often caused by conservative replacements between hydrophobic amino acids. This implies that subtle differences among hydrophobic amino acids are significant for PrPSc structures. Here we analyzed the influence of different hydrophobic residues on the structures of an in-register parallel ß-sheet amyloid of α-synuclein (αSyn) using molecular dynamics (MD) simulation and applied the knowledge from the αSyn amyloid to modeling a local structure of human PrPSc encompassing residues 107-143. We found that mutations equivalent to polymorphisms that cause transmission barriers substantially affect the stabilities of the local structures; for example, the G127V mutation, which makes the host resistant to various human prion diseases, greatly destabilized the local structure of the model amyloid. Our study indicates that subtle differences among hydrophobic side chains can considerably affect the interaction network, including hydrogen bonds, and demonstrates specifically how and in what structures hydrophobic residues can exert unique effects on in-register parallel ß-sheet amyloids.

Synthesis and Biological Evaluation of Novel 2-(Benzofuran-2-yl)-chromone Derivatives for In Vivo Imaging of Prion Deposits in the Brain.

Prion diseases are fatal neurodegenerative disorders caused by the deposition of scrapie prion protein aggregates (PrPSc) in the brain. We previously reported that styrylchromone (SC) and benzofuran (BF) derivatives have potential as imaging probes for PrPSc. To further improve their properties, we designed and synthesized 2-(benzofuran-2-yl)-chromone (BFC) derivatives hybridized with SC and BF backbones as novel single-photon emission computed tomography probes for the detection of cerebral PrPSc deposits. Recombinant mouse prion protein (rMoPrP) aggregates and mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice were used to evaluate the binding properties of BFC derivatives to PrPSc. The BFC derivatives exhibited high binding affinities (equilibrium dissociation constant [Kd] = 22.6-47.7 nM) for rMoPrP aggregates. All BFC derivatives showed remarkable selectivity against amyloid beta aggregates. Fluorescence microscopy confirmed that the fluorescence signals of the BFC derivatives corresponded to the antibody-positive deposits of PrPSc in mBSE-infected mouse brains. Among the BFC derivatives, [125I]BFC-OMe and [125I]BFC-NH2 exhibited high brain uptake and favorable washout from the mouse brain. In vitro autoradiography demonstrated that the distribution of [125I]BFC-OMe in the brain tissues of mBSE-infected mice was colocalized with PrPSc deposits. Taken together, BFC derivatives appear to be promising prion imaging probes.

Synthesis and Characterization of Hydroxyethylamino- and Pyridyl-Substituted 2-Vinyl Chromone Derivatives for Detection of Cerebral Abnormal Prion Protein Deposits.

Prion diseases are fatal neurodegenerative diseases characterized by the deposition of abnormal prion protein aggregates (PrPSc) in the brain. In this study, we developed hydroxyethylamino-substituted styrylchromone (SC) and 2-(2-(pyridin-3-yl)vinyl)-4H-chromen-4-one (VPC) derivatives for single-photon emission computed tomography (SPECT) imaging of PrPSc deposits in the brain. The binding affinity of these compounds was evaluated using recombinant mouse prion protein (rMoPrP) aggregates, which resulted in the inhibition constant (Ki) value of 61.5 and 88.0 nM for hydroxyethyl derivative, (E)-2-(4-((2-hydroxyethyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NHEtOH) and (E)-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NMeEtOH), respectively. However, none of the VPC derivatives showed binding affinity for the rMoPrP aggregates. Fluorescent imaging demonstrated that the accumulation pattern of SC-NHEtOH matched with the presence of PrPSc in the brain slices from mouse-adapted bovine spongiform encephalopathy-infected mice. A biodistribution study of normal mice indicated low initial brain uptake of [125I]SC-NHEtOH (0.88% injected dose/g (% ID/g) at 2 min) despite favorable washout from the brain (0.26% ID/g, at 180 min) was displayed. [125I]SC-NHEtOH exhibited binding affinities to both artificial prion aggregates as well as prion deposits in the brain. However, significant improvement in the binding affinity for PrPSc and blood-brain barrier permeability is necessary for the development of successful in vivo imaging probes for the detection of cerebral PrPSc in the brain.

Lead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction.

Influenza viruses are responsible for contagious respiratory illnesses in humans and cause seasonal epidemics and occasional pandemics worldwide. Previously, we identified a quinolinone derivative PA-49, which inhibited the influenza virus RNA-dependent RNA polymerase (RdRp) by targeting PA-PB1 interaction. This paper reports the structure optimization of PA-49, which resulted in the identification of 3-((dibenzylamino)methyl)quinolinone derivatives with more potent anti-influenza virus activity. During the optimization, the hit compound 89, which was more active than PA-49, was identified. Further optimization and scaffold hopping of 89 led to the most potent compounds 100 and a 1,8-naphthyridinone derivative 118, respectively. We conclusively determined that compounds 100 and 118 suppressed the replication of influenza virus and exhibited anti-influenza virus activity against both influenza virus types A and B in the range of 50% effective concentration (EC50) = 0.061-0.226 µM with low toxicity (50% cytotoxic concentration (CC50) >10 µM).

Stem Cell Therapy for Acute/Subacute Ischemic Stroke with a Focus on Intraarterial Stem Cell Transplantation: From Basic Research to Clinical Trials.

Stem cell therapy for ischemic stroke holds great promise for the treatment of neurological impairment and has moved from the laboratory into early clinical trials. The mechanism of action of stem cell therapy includes the bystander effect and cell replacement. The bystander effect plays an important role in the acute to subacute phase, and cell replacement plays an important role in the subacute to chronic phase. Intraarterial (IA) transplantation is less invasive than intraparenchymal transplantation and can provide more cells in the affected brain region than intravenous transplantation. However, transplanted cell migration was reported to be insufficient, and few transplanted cells were retained in the brain for an extended period. Therefore, the bystander effect was considered the main mechanism of action of IA stem cell transplantation. In most clinical trials, IA transplantation was performed during the acute and subacute phases. Although clinical trials of IA transplantation demonstrated safety, they did not demonstrate satisfactory efficacy in improving patient outcomes. To increase efficacy, increased migration of transplanted cells and production of long surviving and effective stem cells would be crucial. Given the lack of knowledge on this subject, we review and summarize the mechanisms of action of transplanted stem cells and recent advancements in preclinical and clinical studies to provide information and guidance for further advancement of acute/subacute phase IA stem cell transplantation therapy for ischemic stroke.

Development of α-Synuclein Real-Time Quaking-Induced Conversion as a Diagnostic Method for α-Synucleinopathies.

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are characterized by aggregation of abnormal α-synuclein (α-syn) and collectively referred to as α-synucleinopathy. Because these diseases have different prognoses and treatments, it is desirable to diagnose them early and accurately. However, it is difficult to accurately diagnose these diseases by clinical symptoms because symptoms such as muscle rigidity, postural dysreflexia, and dementia sometimes overlap among these diseases. The process of conformational conversion and aggregation of α-syn has been thought similar to that of abnormal prion proteins that cause prion diseases. In recent years, in vitro conversion methods, such as real-time quaking-induced conversion (RT-QuIC), have been developed. This method has succeeded in amplifying and detecting trace amounts of abnormal prion proteins in tissues and central spinal fluid of patients by inducing conversion of recombinant prion proteins via shaking. Additionally, it has been used for antemortem diagnosis of prion diseases. Recently, aggregated α-syn has also been amplified and detected in patients by applying this method and many clinical studies have examined diagnosis using tissues or cerebral spinal fluid from patients. In this review, we discuss the utility and problems of α-syn RT-QuIC for antemortem diagnosis of α-synucleinopathies.

Formalin RT-QuIC assay detects prion-seeding activity in formalin-fixed brain samples from sporadic Creutzfeldt-Jakob disease patients.

BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.

Abnormal prion protein deposits with high seeding activities in the skeletal muscle, femoral nerve, and scalp of an autopsied case of sporadic Creutzfeldt-Jakob disease.

We report the general autopsy findings of abnormal prion protein (PrP) deposits with their seeding activities, as assessed by the real-time quaking-induced conversion (RT-QuIC) method, in a 72-year-old female patient with sporadic Creutzfeldt-Jakob disease (sCJD). At 68 years of age, she presented with gait disturbance and visual disorders. Electroencephalography showed periodic synchronous discharge. Myoclonus was also observed. A genetic test revealed that PRNP codon 129 was methionine/methionine (MM). She died of pneumonia three years and four months after disease onset, and a general autopsy was performed. The brain weighed 650 g and appeared markedly atrophic. Immunohistochemistry for PrP revealed synaptic PrP deposits and coarse PrP deposits in the cerebral cortices, basal ganglia, cerebellum, and brainstem. Western blot analysis identified type 1 proteinase-K-resistant PrP in frontal cortex samples. PrP deposits were also observed in systemic organs, including the femoral nerve, psoas major muscle, abdominal skin, adrenal medulla, zona reticularis of the adrenal gland, islet cells of the pancreas, and thyroid gland. The RT-QuIC method revealed positive seeding activities in all examined organs, including the frontal cortex, femoral nerve, psoas major muscle, scalp, abdominal skin, adrenal gland, pancreas, and thyroid gland. The following 50% seeding dose (SD50 ) values were 9.5 (frontal cortex); 8 ± 0.53 (femoral nerve); 7 ± 0.53 (psoas major muscle); and 7.88 ± 0.17 (scalp). The SD50 values for the adrenal gland, dermis, pancreas, and thyroid gland were 6.12 ± 0.53, 5.25, 4.75, and 4.5, respectively. PrP deposits in general organs may be associated with long-term disease duration. This case indicated the necessity for general autopsies in sCJD cases to establish strict infection control procedures for surgical treatment and to examine certain organs.

Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro.

Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation (LLPS) in the absence of PrPSc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by ß-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid-solid phase transition could be the intermediate steps in the conversion of prions.

Biomarkers and diagnostic guidelines for sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease is a fatal neurodegenerative disease caused by misfolded prion proteins (PrPSc). Effective therapeutics are currently not available and accurate diagnosis can be challenging. Clinical diagnostic criteria use a combination of characteristic neuropsychiatric symptoms, CSF proteins 14-3-3, MRI, and EEG. Supportive biomarkers, such as high CSF total tau, could aid the diagnostic process. However, discordant studies have led to controversies about the clinical value of some established surrogate biomarkers. Development and clinical application of disease-specific protein aggregation and amplification assays, such as real-time quaking induced conversion (RT-QuIC), have constituted major breakthroughs for the confident pre-mortem diagnosis of sporadic Creutzfeldt-Jakob disease. Updated criteria for the diagnosis of sporadic Creutzfeldt-Jakob disease, including application of RT-QuIC, should improve early clinical confirmation, surveillance, assessment of PrPSc seeding activity in different tissues, and trial monitoring. Moreover, emerging blood-based, prognostic, and potentially pre-symptomatic biomarker candidates are under investigation.

Dextran sulphate inhibits an association of prions with plasma membrane at the early phase of infection.

The defining characteristic of prion diseases is conversion of a cellular prion protein (PrPC) to an abnormal prion protein (PrPSc). The exogenous attachment of PrPSc to the surface of a target cell is critical for infection. However, the initial interaction of PrPSc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrPSc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22 L-infected brain homogenates and revealed that PrPSc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrPC-deficient hippocampus cell lines. We also demonstrate here that PrPSc from 22 L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrPSc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.

Generation, optimization and characterization of novel anti-prion compounds.

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.

Feasibility studies of radioiodinated pyridyl benzofuran derivatives as potential SPECT imaging agents for prion deposits in the brain.

INTRODUCTION: Prion diseases are fatal neurodegenerative disorders caused by the deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to evaluate the use of iodinated pyridyl benzofuran (IPBF) derivatives as single-photon emission computed tomography (SPECT) probes for the detection of cerebral PrPSc deposits. METHODS: In vitro binding assays of IPBF derivatives were carried out in the recombinant mouse prion protein (rMoPrP) and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. SPECT imaging of 5-(5-[123I]iodobenzofuran-2-yl)-N-methylpyridin-2-amine ([123I]IPBF-NHMe) was performed on mBSE-infected and mock-infected mice. RESULTS: Fluorescence microscopy results showed that fluorescence signals of IPBF derivatives corresponded to the thioflavin-T positive amyloid deposits of PrPSc in the brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. Among the IPBF derivatives, 5-(5-iodobenzofuran-2-yl)-N-methylpyridin-2-amine (IPBF-NHMe) exhibited the highest binding affinity to the recombinant mouse prion protein (rMoPrP) aggregates with a Ki of 14.3 nM. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that the [123I]IPBF-NHMe distribution in brain tissues of mBSE-infected mice co-localized with PrPSc deposits. CONCLUSION: [123I]IPBF-NHMe appears to be a prospective SPECT tracer for monitoring prion deposits in living brain tissues.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice.

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.

Difference in driver gene expression patterns between perihilar and peripheral intrahepatic cholangiocarcinoma in an experimental mouse model.

BACKGROUND: The prognosis of intrahepatic cholangiocarcinoma (ICC) is based on tumor localization; however, the mechanism remains unknown. Therefore, we investigated the biological characteristics of perihilar and peripheral ICC in a mouse model. METHODS: The model was established by the administration of three oncogenic plasmids harboring myristoylated AKT, mutated human YAP, and pCMV-Sleeping Beauty into the mice. The perihilar and peripheral ICC tumors that developed in the same mouse were assessed for the expression of cell adhesion factors and driver genes with immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The perihilar ICC tumors were irregularly shaped, whereas the peripheral tumors were mostly circular, similar to the differences found in patients. Alpha-smooth muscle actin was strongly expressed in the perihilar tumors at 10 weeks, and vimentin expression was significantly up-regulated in the perihilar ICC at 14 weeks. Fgfr2 level significantly increased in peripheral ICC at 10 weeks, whereas Idh2 expression was up-regulated in perihilar ICC. CONCLUSIONS: Despite diffuse injection of oncogenic plasmid, expression of driver genes and oncogenes in ICC tumor cells differs depending on the tumor localization, resulting in changes in epithelial-mesenchymal transition, which may explain the different outcomes of patients with peripheral and perihilar ICC.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants.

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs.