TMV-CP based rational design and discovery of α-Amide phosphate derivatives as anti plant viral agents.

The tobacco mosaic virus coat protein (TMV-CP) is indispensable for the virus's replication, movement and transmission, as well as for the host plant's immune system to recognize it. It constitutes the outermost layer of the virus particle, and serves as an essential component of the virus structure. TMV-CP is essential for initiating and extending viral assembly, playing a crucial role in the self-assembly process of Tobacco Mosaic Virus (TMV). This research employed TMV-CP as a primary target for virtual screening, from which a library of 43,417 compounds was sourced and SH-05 was chosen as the lead compound. Consequently, a series of α-amide phosphate derivatives were designed and synthesized, exhibiting remarkable anti-TMV efficacy. The synthesized compounds were found to be beneficial in treating TMV, with compound 3g displaying a slightly better curative effect than Ningnanmycin (NNM) (EC50 = 304.54 µg/mL) at an EC50 of 291.9 µg/mL. Additionally, 3g exhibited comparable inactivation activity (EC50 = 63.2 µg/mL) to NNM (EC50 = 67.5 µg/mL) and similar protective activity (EC50 = 228.9 µg/mL) to NNM (EC50 = 219.7 µg/mL). Microscale thermal analysis revealed that the binding of 3g (Kd = 4.5 ± 1.9 µM) to TMV-CP showed the same level with NNM (Kd = 5.5 ± 2.6 µM). Results from transmission electron microscopy indicated that 3g could disrupt the structure of TMV virus particles. The toxicity prediction indicated that 3g was low toxicity. Molecular docking showed that 3g interacted with TMV-CP through hydrogen bond, attractive charge interaction and π-Cation interaction. This research provided a novel α-amide phosphate structure target TMV-CP, which may help the discovery of new anti-TMV agents in the future.

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening.

Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.

Hsp70 Inhibits the Replication of Fowl Adenovirus Serotype 4 by Suppressing Viral Hexon with the Assistance of DnaJC7.

Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. IMPORTANCE FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.

Discovery of aminothiazole derivatives as novel human enterovirus A71 capsid protein inhibitors.

Enterovirus A71 (EV-A71), one of the major pathogens that causes hand, foot and mouth disease (HFMD), has seriously threatened the health and safety of young children. In this study, aminothiazole derivatives were synthesized and screened against EV-A71 in Rhabdomyosarcoma (RD) cells. The best compound (12s), with a biphenyl group, showed activity against EV-A71 (EC50: 0.27 µM) but also against a series of different human enteroviruses without significant cytotoxicity (CC50 > 56.2 µM). Mechanistic studies including time-of-drug-addition assays, viral entry assays and microscale thermophoresis (MST) experiments, showed that 12s binds to EV-A71 capsid and blocks the binding between the viral protein VP1 and the relevant human scavenger receptor class B member 2 (hSCARB2).

Targeting the Virus Capsid as a Tool to Fight RNA Viruses.

Several strategies have been developed to fight viral infections, not only in humans but also in animals and plants. Some of them are based on the development of efficient vaccines, to target the virus by developed antibodies, others focus on finding antiviral compounds with activities that inhibit selected virus replication steps. Currently, there is an increasing number of antiviral drugs on the market; however, some have unpleasant side effects, are toxic to cells, or the viruses quickly develop resistance to them. As the current situation shows, the combination of multiple antiviral strategies or the combination of the use of various compounds within one strategy is very important. The most desirable are combinations of drugs that inhibit different steps in the virus life cycle. This is an important issue especially for RNA viruses, which replicate their genomes using error-prone RNA polymerases and rapidly develop mutants resistant to applied antiviral compounds. Here, we focus on compounds targeting viral structural capsid proteins, thereby inhibiting virus assembly or disassembly, virus binding to cellular receptors, or acting by inhibiting other virus replication mechanisms. This review is an update of existing papers on a similar topic, by focusing on the most recent advances in the rapidly evolving research of compounds targeting capsid proteins of RNA viruses.

Design, synthesis, and mechanistic investigations of phenylalanine derivatives containing a benzothiazole moiety as HIV-1 capsid inhibitors with improved metabolic stability.

Further clinical development of PF74, a lead compound targeting HIV-1 capsid, is impeded by low antiviral activity and inferior metabolic stability. By modifying the benzene (region I) and indole of PF74, we identified two potent compounds (7m and 7u) with significantly improved metabolic stability. Compared to PF74, 7u displayed greater metabolic stability in human liver microsomes (HLMs) with half-life (t1/2) 109-fold that of PF74. Moreover, mechanism of action (MOA) studies demonstrated that 7m and 7u effectively mirrored the MOA of compounds that interact within the PF74 interprotomer pocket, showing direct and robust interactions with recombinant CA, and 7u displaying antiviral effects in both the early and late stages of HIV-1 replication. Furthermore, MD simulation corroborated that 7u was bound to the PF74 binding site, and the results of the online molinspiration software predicted that 7m and 7u had desirable physicochemical properties. Unexpectedly, this series of compounds exhibited better antiviral activity than PF74 against HIV-2, represented by compound 7m whose anti-HIV-2 activity was almost 5 times increased potency over PF74. Therefore, we have rationally redesigned the PF74 chemotype to inhibitors with novel structures and enhanced metabolic stability in this study. We hope that these new compounds can serve as a blueprint for developing a new generation of HIV treatment regimens.

Identification of (6S)-cyclopropyl-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxamines as new HBV capsid assembly modulators.

GYH2-18 is a type II HBV CAM with 6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxamine (DPPC) skeleton discovered by Roche INC. A series of GYH2-18 derivatives were designed, synthesized and evaluated for their anti-HBV activity. Two compounds 2f and 3k exhibited excellent anti-HBV activity, low cytotoxicity and accepted oral PK profiles. Chiral separation of 2f and 3k was conducted successfully, and (6S)-cyclopropyl DPPC isomers 2f-1, 2f-3, 3k-1 and 3k-3 were identified to be much more active than the corresponding (6R)-ones. The preliminary structure-activity relationship, particle gel assay and molecular modeling studies were also discussed, which provide useful indications for guiding the further rational design of new (6S)-cyclopropyl DPPC analogues.

Long-acting capsid inhibitor protects macaques from repeat SHIV challenges.

Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian-human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg-1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans.

Searching for drug leads targeted to the hydrophobic cleft of dengue virus capsid protein.

We synthesised and screened 18 aromatic derivatives of guanylhydrazones and oximes aromatic for their capacity to bind to dengue virus capsid protein (DENVC). The intended therapeutic target was the hydrophobic cleft of DENVC, which is a region responsible for its anchoring in lipid droplets in the infected cells. The inhibition of this process completely suppresses virus infectivity. Using NMR, we describe five compounds able to bind to the α1-α2 interface in the hydrophobic cleft. Saturation transfer difference experiments showed that the aromatic protons of the ligands are important for the interaction with DENVC. Fluorescence binding isotherms indicated that the selected compounds bind at micromolar affinities, possibly leading to binding-induced conformational changes. NMR-derived docking calculations of ligands showed that they position similarly in the hydrophobic cleft. Cytotoxicity experiments and calculations of in silico drug properties suggest that these compounds may be promising candidates in the search for antivirals targeting DENVC.

Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure, Mode-of-Action and Efficacy.

Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.

Oxadiazepinone HBV capsid assembly modulators.

The HBV core protein serves multiple essential functions in the viral life cycle that enable chronic HBV infection to persist, and as such, represents a promising drug target. Modulation of the HBV capsid assembly has shown efficacy in early clinical trials through use of small molecule capsid assembly modulators (CAMs). Herein is described the evolution and SAR of a novel pyrazolo piperidine lead series into advanced oxadiazepinone HBV CAMs.

Design, synthesis, and mechanism study of dimerized phenylalanine derivatives as novel HIV-1 capsid inhibitors.

HIV-1 capsid (CA) plays indispensable and multiple roles in the life cycle of HIV-1, become an attractive target in antiviral therapy. Herein, we report the design, synthesis, and mechanism study of a novel series of dimerized phenylalanine derivatives as HIV-1 capsid inhibitors using 2-piperazineone or 2,5-piperazinedione as a linker. The structure-activity relationship (SAR) indicated that dimerized phenylalanines were more potent than monomers of the same chemotype. Further, the inclusion of fluorine substituted phenylalanine and methoxyl substituted aniline was found to be beneficial for antiviral activity. From the synthesized series, Q-c4 was found to be the most potent compound with an EC50 value of 0.57 µM, comparable to PF74. Interestingly, Q-c4 demonstrated a slightly higher affinity to the CA monomer than the CA hexamer, commensurate with its more significant effect in the late-stage of the HIV-1 lifecycle. Competitive SPR experiments with peptides from CPSF6 and NUP153 revealed that Q-c4 binds to the interprotomer pocket of hexameric CA as designed. Single-round infection assays showed that Q-c4 interferes with the HIV-1 life cycle in a dual-stage manner, affecting both pre-and post-integration. Stability assays in human plasma and human liver microsomes indicated that although Q-c4 has improved stability over PF74, this kind of inhibitor still requires further optimization. And the results of the online molinspiration software predicted that Q-c4 has desirable physicochemical properties but some properties still have some violation from the Lipinski rule of five. Overall, the dimerized phenylalanines are promising novel platforms for developing future HIV-1 CA inhibitors with considerable potential for optimization.

Design, synthesis, and antiviral activity of phenylalanine derivatives as HIV-1 capsid inhibitors.

The HIV-1 Capsid (CA) is considered as a promising target for the development of potent antiviral drugs, due to its multiple roles during the viral life cycle. Herein, we report the design, synthesis, and antiviral activity evaluation of series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors. Among them, 4-methoxy-N-methylaniline substituted phenylalanine (II-13c) and indolin-5-amine substituted phenylalanine (V-25i) displayed exceptional anti-HIV-1 activity with the EC50 value of 5.14 and 2.57 µM respectively, which is slightly weaker than that of lead compound PF-74 (EC50 = 0.42 µM). Besides, surface plasmon resonance (SPR) binding assay demonstrated II-13c and V-25i prefer to combine with CA hexamer rather than monomer, which is similar to PF-74. Subsequently, molecular dynamics simulation (MD) revealed potential interactions between representative compounds with HIV-1 CA hexamer. Overall, this work laid a solid foundation for further structural optimization to discover novel promising HIV-1 CA inhibitors.

Design, synthesis and evaluation of heteroaryldihydropyrimidine analogues bearing spiro ring as hepatitis B virus capsid protein inhibitors.

GLS4, a potent antiviral drug candidate, has been widely studied and entered into phase II clinical trials. Nevertheless, the therapeutic application of GLS4 is limited due to poor water solubility, short half-life, and low bioavailability. In order to improve the hydrophilicity and pharmacokinetic (PK) properties of GLS4, herein, we retained the dominant fragments, and used a scaffold hopping strategy to replace the easily metabolized morpholine ring of GLS4 with diverse sizes of spiro rings consisting of hydrogen bond donor and acceptor substituents. Potent in vitroanti-HBV activity and low cytotoxicity were observed for compound 4r (EC50 = 0.20 ± 0.00 µM, CC50 > 87.03 µM), which was more potent than the positive control lamivudine (EC50 = 0.37 ± 0.04 µM, CC50 > 100.00 µM) in this assay and was about a quarter as effective as GLS4 (EC50 = 0.045 ± 0.01 µM, CC50 > 99.20 µM). Preliminary structure-activity relationship (SAR) analysis and molecular docking studies were carried out to explore potential interactions and binding mode between compounds and target protein. In terms of the physicochemical properties, 4r was predicted to be consistent with the rule-of-five, which means 4r may have favourable absorption and permeation. Finally, ADMET and PK characteristics of 4r and GLS4 were predicted to be comparable in most aspects, implying that the two compounds may have similar profiles in vivo.

Identification of a Potential Inhibitor of the FIV p24 Capsid Protein and Characterization of Its Binding Site.

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.

Antiviral activity of Mulberroside C against enterovirus A71 in vitro and in vivo.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease which seriously threatens young children's health and lives. However, there is no effective therapy currently available for treating these infections. Therefore, effective drugs to prevent and treat EV-A71 infections are urgently needed. Here, we identified Mulberroside C potently against the proliferation of EV-A71. The in-vitro anti-EV-A71 activity of Mulberroside C was assessed by cytopathic effect inhibition and viral plaque reduction assays, and the results showed that Mulberroside C significantly inhibited EV-A71 infection. The downstream assays affirmed that Mulberroside C inhibited viral protein and RNA synthesis. Furthermore, Mulberroside C effectively reduced clinical symptoms in EV-A71 infected mice and reduced mortality at higher concentrations. The mechanism study indicated that Mulberroside C bound to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and genome release. Taken together, our study indicated that Mulberroside C could be a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

An insight on medicinal aspects of novel HIV-1 capsid protein inhibitors.

A capsid is the protein shell of a virus, encircling its genetic material. The HIV capsid is erected from a single protein, known as capsid protein. The capsid of HIV-1 significantly involved in many processes of the virus life cycle, which makes it as a novel target for the new inhibitors. Recently many novel HIV-1 inhibitors binding to capsid proteins have been reported successfully. Most of these inhibitors can inhibit or accelerate the disassembly or assembly of the capsid, and some of them can inhibit reverse transcription. Unfortunately, none of them are currently approved by U.S. FDA. However, GS-6207, an inhibitor binds to the NTD-CTD interface with potent antiviral activity and the long metabolic cycle, is expected to be the first approved drug targeting HIV-1 capsid. Herein, we provide a concise report focusing on the recent prospective of HIV-1 capsid inhibitors in medicinal chemistry in order to enlighten drug design.

Rapid Optimization of the Metabolic Stability of a Human Immunodeficiency Virus Type-1 Capsid Inhibitor Using a Multistep Computational Workflow.

Poor metabolic stability of the human immunodeficiency virus type-1 (HIV-1) capsid (CA) inhibitor PF-74 is a major concern in its development toward clinical use. To improve on the metabolic stability, we employed a novel multistep computationally driven workflow, which facilitated the rapid design of improved PF-74 analogs in an efficient manner. Using this workflow, we designed three compounds that interact specifically with the CA interprotomer pocket, inhibit HIV-1 infection, and demonstrate enantiomeric preference. Moreover, using this workflow, we were able to increase the metabolic stability 204-fold in comparison to PF-74 in only three analog steps. These results demonstrate our ability to rapidly design CA compounds using a novel computational workflow that has improved metabolic stability over the parental compound. This workflow can be further applied to the redesign of PF-74 and other promising inhibitors with a stability shortfall.

Discovery of novel HBV capsid assembly modulators by structure-based virtual screening and bioassays.

HBV capsid assembly has been regarded as an attractive potential target for anti-HBV therapy. In this study, we discovery the Novel HBV capsid assembly modulators (CAMs) through structure-based virtual screening and bioassays. A total of 16 structurally diverse compounds were purchased and assayed, including three compounds with inhibition rate > 50% at 20 µM. Further lead optimization based on the most potent compound II-1-7 (EC50 = 5.6 ± 0.1 µM) were performed by using substructure searching strategy, resulting in compound II-2-9 with an EC50 value of 1.8 ± 0.6 µM. In bimolecular fluorescence complementation (BiFC) assay, compound II-2-9 inhibited the HBV by disrupting the HBV capsid interactions. In summary, this study provides a highly efficient way to discover novel CAMs, and 2-aryl-4-quinolyl amide derivatives could serve as the starting point for development of novel anti-HBV drugs.

Identification of a new class of HBV capsid assembly modulator.

The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly has shown efficacy in clinical trials. Herein is described the identification and hit to lead SAR of a novel series of pyrazolo piperidine HBV capsid assembly modulators.