Botulinum Toxin for Pediatric Migraine: A Retrospective Multisite Cohort Study.

BACKGROUND: Onabotulinum toxin A is effective in adult chronic migraine, but the efficacy is not well established in adolescent patients. The objective of this study is to describe the safety and efficacy of onabotulinum toxin A and incobotulinum toxin A for adolescent chronic migraine headache. METHODS: We performed a chart review of adolescents who received onabotulinum toxin A or incobotulinum toxin A for headache prevention. Demographic information and baseline headache characteristics were collected. The primary end point was a 50% reduction in headache frequency. Secondary outcome measures included reduction in headache frequency, repeat appointments for injections, reduction in other migraine medications, and adverse events. RESULTS: We included 51 adolescents who received at least one injection of either incobotulinum toxin A or onabotulinum toxin A for chronic migraine. Mean age at first dose was 16.0 (1.1; 13 to 17), (S.D. and range). Patients averaged 24.0 headache days per month (7.6; 4 to 28), (S.D. and range) before injection. In addition, 36 of the 51 adolescents (71%) were experiencing continuous headaches. Thirty-five (69%) adolescents had experienced 50% reduction in headache days by the time of first follow-up, which occurred on average at 16.6 weeks from initial injection (11.5; 2 to 55.7) (S.D. and range). Adolescents reported an average decrease of 13.1 headaches days per month. Only two adolescents reported side effects (4%), which were neck soreness and headache following injection. CONCLUSIONS: Botulinum toxin had better efficacy in our adolescent migraine population than has been demonstrated in other studies.

Peptidomimetic α-Acyloxymethylketone Warheads with Six-Membered Lactam P1 Glutamine Mimic: SARS-CoV-2 3CL Protease Inhibition, Coronavirus Antiviral Activity, and in Vitro Biological Stability.

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

SARS-COV-2 recombinant Receptor-Binding-Domain (RBD) induces neutralizing antibodies against variant strains of SARS-CoV-2 and SARS-CoV-1.

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2.

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.

Author Correction: Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication.

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

Comprehensive in vitro characterization of PD-L1 small molecule inhibitors.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Highly bioavailable silibinin nanoparticles inhibit HCV infection.

OBJECTIVE: Silibinin is a flavonolignan that is well established for its robust antiviral activity against HCV infection and has undergone several clinical trials for the management of hepatitis C. Despite its potency, silibinin suffers from poor solubility and bioavailability, restricting its clinical use. To overcome this limitation, we developed highly bioavailable silibinin nanoparticles (SB-NPs) and evaluated their efficiency against HCV infection. DESIGN: SB-NPs were prepared using a nanoemulsification technique and were physicochemically characterised. Infectious HCV culture systems were used to evaluate the influence of SB-NP on the virus life cycle and examine their antioxidant activity against HCV-induced oxidative stress. The safety profiles of SB-NP, in vivo pharmacokinetic studies and antiviral activity against infection of primary human hepatocytes were also assessed. RESULTS: SB-NP consisted of nanoscale spherical particles (<200 nm) encapsulating amorphous silibinin at >97% efficiency and increasing the compound's solubility by >75%. Treatment with SB-NP efficiently restricted HCV cell-to-cell transmission, suggesting that they retained silibinin's robust anti-HCV activity. In addition, SB-NP exerted an antioxidant effect via their free radical scavenging function. Oral administration of SB-NP in rodents produced no apparent in vivo toxicity, and pharmacokinetic studies revealed an enhanced serum level and superior biodistribution to the liver compared with non-modified silibinin. Finally, SB-NP efficiently reduced HCV infection of primary human hepatocytes. CONCLUSIONS: Due to SB-NP's enhanced bioavailability, effective anti-HCV activity and an overall hepatoprotective effect, we suggest that SB-NP may be a cost-effective anti-HCV agent that merits further evaluation for the treatment of hepatitis C.

Saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis C virus entry.

BACKGROUND & AIMS: A vaccine against hepatitis C virus (HCV) is unavailable and cost-effective antivirals that prevent HCV infection and re-infection, such as in the transplant setting, do not exist. In a search for novel and economical prophylactic agents, we examined the antiviral activity of saikosaponins (SSa, SSb2, SSc, and SSd) from Bupleurum kaoi root (BK) as entry inhibitors against HCV infection. METHODS: Infectious HCV culture systems were used to examine the effect of saikosaponins on the complete virus life cycle (entry, RNA replication/translation, and particle production). Antiviral activity against various HCV genotypes, clinical isolates, and infection of primary human hepatocytes were also evaluated. RESULTS: BK and the saikosaponins potently inhibited HCV infection at non-cytotoxic concentrations. These natural agents targeted early steps of the viral life cycle, while leaving replication/translation, egress, and spread relatively unaffected. In particular, we identified SSb2 as an efficient inhibitor of early HCV entry, including neutralization of virus particles, preventing viral attachment, and inhibiting viral entry/fusion. Binding analysis, using soluble viral glycoproteins, demonstrated that SSb2 acted on HCV E2. Moreover, SSb2 inhibited infection by several genotypic strains and prevented binding of serum-derived HCV onto hepatoma cells. Finally, treatment with the compound blocked HCV infection of primary human hepatocytes. CONCLUSIONS: Due to its potency, SSb2 may be of value for development as an antagonist of HCV entry and could be explored as prophylactic treatment during the course of liver transplantation.

Hepatitis C virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication.

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

Rapamycin-mediated suppression of renal cyst expansion in del34 Pkd1-/- mutant mouse embryos: an investigation of the feasibility of renal cyst prevention in the foetus.

AIM: Polycystic kidney disease (PKD) in humans involves kidney cyst expansion beginning in utero. Recessive PKD can result in end-stage renal disease (ESRD) within the first decade, whereas autosomal dominant PKD (ADPKD), caused by mutations in the PKD1 or PKD2 gene, typically leads to ESRD by the fifth decade of life. Inhibition of mTOR signalling was recently found to halt cyst formation in adult ADPKD mice. In contrast, no studies have investigated potential treatments to prevent cyst formation in utero in recessive PKD. Given that homozygous Pkd1 mutant mice exhibit cyst formation in utero, we decided to investigate whether mTOR inhibition in utero ameliorates kidney cyst formation in foetal Pkd1 homozygous mutant mice. METHODS: Pregnant Pkd1(+/-) female mice (mated with Pkd1(+/-) male mice) were treated with rapamycin from E14.5 to E17.5. Foetal kidneys were dissected, genotyped and evaluated by cyst size as well as expression of the developmental marker, Pax2. RESULTS: Numerous cysts were present in Pkd1(-/-) kidneys, which were twice the weight of wild-type kidneys. Cyst size was reduced by a third in rapamycin-treated Pkd1(-/-) kidney sections and kidney mass was reduced to near wild-type levels. However, total cyst number was not reduced compared with control embryos. Pax2 expression and kidney development were unaltered in rapamycin-treated mice but some lethality was observed in Pkd1(-/-) null embryos. CONCLUSION: Rapamycin treatment reduces cyst formation in Pkd1(-/-) mutant mice; therefore, the prevention of kidney cyst expansion in utero by mTOR inhibition is feasible. However, selective rapamycin-associated lethality limits its usefulness as a treatment in utero.

Screening of drugs to counteract human papillomavirus 16 E6 repression of E-cadherin expression.

Persistent infections with certain high-risk human papillomavirus (HPV) types such as 16 and 18 can result in the development of cervical cancer. Neither of the two prophylactic vaccines against HPV16 and 18 that are in current use have any therapeutic efficacy for prevalent HPV infections. Ablative therapy is widely used for the treatment of HPV cervical dysplasia however disease recurrence is a widely recognized problem. Thus there is a continuing need for therapeutic approaches for the treatment of HPV infections. The HPV16 E6 viral oncoprotein represses surface expression of the cellular adhesion molecule, E-cadherin. Reduced E-cadherin expression on HPV-infected keratinocytes is associated with lowered numbers of antigen-presenting Langerhans cells in the infected epidermis, potentially reducing immune surveillance for HPV. Four chemicals reported to up-regulate E-cadherin were screened for their ability to counteract E6 repression of surface E-cadherin. 5-Aza-2'-deoxycytidine (AzaDC), a DNA methyltransferase inhibitor, and Indole-3-carbinol (I3C), reported to increase E-cadherin through a p21(Waf1/Cip1)-dependent mechanism, had low cytotoxicity and increased or restored E-cadherin expression and adhesive function in HPV16 E6 expressing HCT116 cells. Doxorubicin, also known to induce p21(Waf1/Cip1), increased E-cadherin in E6 expressing cells but had some associated cytotoxicity. Tamoxifen, which can restore adhesive function of surface E-cadherin, was ineffective in counteracting E6 repression of E-cadherin. AzaDC and I3C both show potential to restore antigen-presenting cells to HPV infected skin by antagonizing E6 repression of E-cadherin, thereby counteracting an important immune evasion mechanism of HPV16 and reinstating immune function at the infected site.

Functional changes, increased apoptosis, and diminished nuclear factor-kappaB activity of myeloid dendritic cells during chronic hepatitis C infection.

Approximately 70% of patients infected with hepatitis C virus (HCV) develop chronic infections, which have been reported to be caused by impaired specific T-cell responses. Myeloid dendritic cells (mDCs) are important antigen-presenting cells that regulate T-cell responses, however their role during chronic hepatitis C (CHC) is not fully understood. In this study, we found that the ability of mDCs to stimulate T-cell responses was impaired in CHC patients. Furthermore, mDCs from CHC patients underwent apoptosis at a higher rate than mDCs from healthy donors. Nuclear factor-kappaB activity, which is critical for mDC function and apoptosis prevention, was diminished in mDCs from CHC patients. In conclusion, mDCs from CHC patients demonstrated functional changes with increased apoptosis, and diminished nuclear factor-kappaB activity. These changes may contribute to the impaired specific T-cell responses in CHC patients.