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1.
Hepatology ; 2021 Dec 03.
Article in English | MEDLINE | ID: mdl-34860418

ABSTRACT

BACKGROUND AND AIMS: Proof-of-concept studies demonstrated lonafarnib (LNF), a first-in-class oral prenylation inhibitor, efficacy in patients infected with HDV. The lonafarnib with ritonavir for HDV-2 (LOWR-2) study's aim was to identify optimal combination regimens of LNF + ritonavir (RTV) ± pegylated interferon alpha (PEG-IFNα) with efficacy and tolerability for longer-term dosing. Here we report the safety and efficacy at end of treatment for up to 24 weeks. APPROACH AND RESULTS: Fifty-five patients with chronic HDV were consecutively enrolled in an open-label, single-center, phase 2 dose-finding study. There were three main treatment groups: high-dose LNF (LNF ≥ 75 mg by mouth [po] twice daily [bid] + RTV) (n = 19, 12 weeks); all-oral low-dose LNF (LNF 25 or 50 mg po bid + RTV) (n = 24, 24 weeks), and combination low-dose LNF with PEG-IFNα (LNF 25 or 50 mg po bid + RTV + PEG-IFNα) (n = 12, 24 weeks). The primary endpoint, ≥2 log10 decline or < lower limit of quantification of HDV-RNA from baseline at end of treatment, was reached in 46% (6 of 13) and 89% (8 of 9) of patients receiving the all-oral regimen of LNF 50 mg bid + RTV, and combination regimens of LNF (25 or 50 mg bid) + RTV + PEG-IFNα, respectively. In addition, multiple patients experienced well-tolerated transient posttreatment alanine aminotransferase increases, resulting in HDV-RNA negativity and alanine aminotransferase normalization. The proportions of grade 2 and 3 gastrointestinal adverse events in the high-dose versus low-dose groups were 49% (37 of 76) and only 22% (18 of 81), respectively. CONCLUSIONS: LNF, boosted with low-dose RTV, is a promising all-oral therapy, and maximal efficacy is achieved with PEG-IFNα addition. The identified optimal regimens support a phase 3 study of LNF for the treatment of HDV.

2.
Nat Commun ; 12(1): 1967, 2021 03 30.
Article in English | MEDLINE | ID: mdl-33785743

ABSTRACT

Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized, single-blind, placebo-controlled trial (NCT04331899) in 120 outpatients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) within 72 hours of diagnosis could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint). In both the 60 patients receiving Lambda and 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively, and symptom duration did not differ significantly between groups (HR 0.94; 95% CI 0.64 to 1.39). Both Lambda and placebo were well-tolerated, though liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.


Subject(s)
Antiviral Agents/administration & dosage , COVID-19/drug therapy , Interleukins/administration & dosage , Polyethylene Glycols/administration & dosage , Adult , Aged , COVID-19/virology , Female , Humans , Injections, Subcutaneous , Male , Middle Aged , Outpatients , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Single-Blind Method , Treatment Failure , Virus Shedding/drug effects , Young Adult
3.
Lancet Respir Med ; 9(5): 498-510, 2021 05.
Article in English | MEDLINE | ID: mdl-33556319

ABSTRACT

BACKGROUND: To date, only monoclonal antibodies have been shown to be effective for outpatients with COVID-19. Interferon lambda-1 is a type III interferon involved in innate antiviral responses with activity against respiratory pathogens. We aimed to investigate the safety and efficacy of peginterferon lambda in the treatment of outpatients with mild-to-moderate COVID-19. METHODS: In this double-blind, placebo-controlled trial, outpatients with laboratory-confirmed COVID-19 were randomly assigned to a single subcutaneous injection of peginterferon lambda 180 µg or placebo within 7 days of symptom onset or first positive swab if asymptomatic. Participants were randomly assigned (1:1) using a computer-generated randomisation list created with a randomisation schedule in blocks of four. At the time of administration, study nurses received a sealed opaque envelope with the treatment allocation number. The primary endpoint was the proportion of patients who were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on day 7 after the injection, analysed by a χ2 test following an intention-to-treat principle. Prespecified analysis of the primary endpoint, adjusted for baseline viral load, using bivariate logistic regression was done. The trial is now complete. This trial is registered with ClinicalTrials.gov, NCT04354259. FINDINGS: Between May 18, and Sept 4, 2020, we recruited 30 patients per group. The decline in SARS-CoV-2 RNA was greater in those treated with peginterferon lambda than placebo from day 3 onwards, with a difference of 2·42 log copies per mL at day 7 (p=0·0041). By day 7, 24 (80%) participants in the peginterferon lambda group had an undetectable viral load, compared with 19 (63%) in the placebo group (p=0·15). After controlling for baseline viral load, patients in the peginterferon lambda group were more likely to have undetectable virus by day 7 than were those in the placebo group (odds ratio [OR] 4·12 [95% CI 1·15-16·73; p=0·029). Of those with baseline viral load above 106 copies per mL, 15 (79%) of 19 patients in the peginterferon lambda group had undetectable virus on day 7, compared with six (38%) of 16 in the placebo group (OR 6·25 [95% CI 1·49-31·06]; p=0·012). Peginterferon lambda was well tolerated, and adverse events were similar between groups with mild and transient aminotransferase, concentration increases more frequently observed in the peginterferon lambda group. Two individuals met the threshold of grade 3 increase, one in each group, and no other grade 3 or 4 laboratory adverse events were reported. INTERPRETATION: Peginterferon lambda accelerated viral decline in outpatients with COVID-19, increasing the proportion of patients with viral clearance by day 7, particularly in those with high baseline viral load. Peginterferon lambda has potential to prevent clinical deterioration and shorten duration of viral shedding. FUNDING: The Toronto COVID-19 Action Initiative, University of Toronto, and the Ontario First COVID-19 Rapid Research Fund, Toronto General & Western Hospital Foundation.


Subject(s)
Ambulatory Care/methods , COVID-19 , Interleukins , Polyethylene Glycols , SARS-CoV-2 , Viral Load/drug effects , Virus Shedding/drug effects , Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19/immunology , Double-Blind Method , Drug Monitoring/methods , Female , Humans , Intention to Treat Analysis , Interleukins/administration & dosage , Interleukins/adverse effects , Male , Middle Aged , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/adverse effects , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Severity of Illness Index , Treatment Outcome
5.
Nature ; 586(7830): 560-566, 2020 10.
Article in English | MEDLINE | ID: mdl-32854108

ABSTRACT

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.


Subject(s)
Betacoronavirus , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Disease Models, Animal , Interferons/pharmacology , Interferons/therapeutic use , Interleukins/pharmacology , Interleukins/therapeutic use , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Aging/immunology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/drug effects , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Female , Forkhead Transcription Factors/genetics , Humans , Interferon-alpha/administration & dosage , Interferon-alpha/pharmacology , Interferon-alpha/therapeutic use , Interferons/administration & dosage , Interleukins/administration & dosage , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Models, Molecular , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/genetics , Pneumonia, Viral/immunology , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2
6.
bioRxiv ; 2020 May 07.
Article in English | MEDLINE | ID: mdl-32511406

ABSTRACT

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs). SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. We used reverse genetics to remodel the S and mACE2 binding interface resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro , and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of IFN lambda-1a treatment in human COVID-19 infections.

7.
Sci Transl Med ; 12(527)2020 01 22.
Article in English | MEDLINE | ID: mdl-31969487

ABSTRACT

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.


Subject(s)
Adenocarcinoma of Lung/metabolism , Chromosomes, Human, Pair 1/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Adenocarcinoma of Lung/genetics , Animals , Chromosomes, Human, Pair 1/genetics , Enzyme-Linked Immunosorbent Assay , Golgi Apparatus/metabolism , Humans , In Vitro Techniques , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , X-Ray Microtomography
8.
Diagn Microbiol Infect Dis ; 92(2): 112-117, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29941366

ABSTRACT

In this study, a real-time reverse transcription-polymerase chain reaction (real time RT-PCR) assay targeting 2 genetic segments was established to detect HDV RNA. Utilizing the World Health Organization International Standard for Hepatitis D Virus RNA, the lower limit of detection was 575 IU/mL, and the linearity of quantification ranged from 575,000 IU/mL to 575 IU/mL. 384 HBsAg-positive samples collected from China were tested by this method and HDV antibody detection. Eleven samples were positive for anti-HDV IgG which may persist after HDV resolution, 6 samples were HDV RNA positive, and 5 samples were positive for anti-HDV IgM. This assay showed more sensitivity than the detection of anti-HDV IgM. These data demonstrate that the real-time RT-PCR assay for HDV RNA could be implemented in the clinical detection of HDV infection in chronic HBV-infected patients in China.


Subject(s)
Hepatitis Antibodies/immunology , Hepatitis D/diagnosis , Hepatitis Delta Virus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Adult , China , Female , Genotype , Hepatitis D/virology , Hepatitis Delta Virus/genetics , Hepatitis Delta Virus/immunology , Humans , Male , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral Load
9.
Anal Chem ; 88(10): 5042-5, 2016 05 17.
Article in English | MEDLINE | ID: mdl-27118725

ABSTRACT

The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIß (PI4Kß) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kß followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI(4,5)P2 or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI(4,5)P2 generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kß by a small molecule inhibitor was also quantitatively assessed, yielding an EC50 value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides.


Subject(s)
1-Phosphatidylinositol 4-Kinase/chemistry , Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , 1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Membrane Proteins/antagonists & inhibitors , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol Phosphates/chemistry , Quartz Crystal Microbalance Techniques
10.
Bioorg Med Chem Lett ; 20(14): 4060-4, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20541404

ABSTRACT

A pyridazin-4-one fragment 4 (hCatS IC(50)=170 microM) discovered through Tethering was modeled into cathepsin S and predicted to overlap in S2 with the tetrahydropyridinepyrazole core of a previously disclosed series of CatS inhibitors. This fragment served as a template to design pyridazin-3-one 12 (hCatS IC(50)=430 nM), which also incorporates P3 and P5 binding elements. A crystal structure of 12 bound to Cys25Ser CatS led to the synthesis of the potent diazinone isomers 22 (hCatS IC(50)=60 nM) and 27 (hCatS IC(50)=40 nM).


Subject(s)
Cathepsins/antagonists & inhibitors , Protease Inhibitors/chemistry , Pyrazoles/chemistry , Crystallography, X-Ray , Models, Molecular , Structure-Activity Relationship
11.
12.
Bioorg Med Chem Lett ; 18(21): 5763-5, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18842409

ABSTRACT

The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Compound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and higher bioavailability in mice.


Subject(s)
Oxazoles/chemistry , Oxazoles/pharmacokinetics , Thiazoles/chemistry , Thiazoles/pharmacokinetics , Animals , Biological Availability , Mice , Permeability
13.
Bioorg Med Chem Lett ; 18(20): 5648-52, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18793847

ABSTRACT

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyridines/chemical synthesis , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Cell Cycle , Crystallography, X-Ray , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , Phenotype , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Structure-Activity Relationship
14.
Science ; 310(5750): 1022-5, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16284179

ABSTRACT

We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.


Subject(s)
Indoles/chemistry , Indoles/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/chemistry , Biotinylation , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Dimerization , Fluorescence , Hydrogen/chemistry , Hydrophobic and Hydrophilic Interactions , Indoles/chemical synthesis , Kinetics , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Conformation , Protein Subunits/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolism
16.
Nat Biotechnol ; 21(3): 308-14, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12563278

ABSTRACT

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.


Subject(s)
Caspase Inhibitors , Caspases/chemistry , Combinatorial Chemistry Techniques/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Jurkat Cells/drug effects , Apoptosis/drug effects , Caspase 3 , Caspases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/classification , Humans , Jurkat Cells/metabolism , Mass Spectrometry/methods , Models, Molecular , Peptide Library
17.
J Med Chem ; 45(23): 5005-22, 2002 Nov 07.
Article in English | MEDLINE | ID: mdl-12408711

ABSTRACT

The design, synthesis, and in vitro activities of a series of potent and selective small-molecule inhibitors of caspase-3 are described. From extended tethering, a salicylic acid fragment was identified as having binding affinity for the S(4) pocket of caspase-3. X-ray crystallography and molecular modeling of the initial tethering hit resulted in the synthesis of 4, which reversibly inhibited caspase-3 with a K(i) = 40 nM. Further optimization led to the identification of a series of potent and selective inhibitors with K(i) values in the 20-50 nM range. One of the most potent compounds in this series, 66b, inhibited caspase-3 with a K(i) = 20 nM and selectivity of 8-500-fold for caspase-3 vs a panel of seven caspases (1, 2, and 4-8). A high-resolution X-ray cocrystal structure of 4 and 66b supports the predicted binding modes of our compounds with caspase-3.


Subject(s)
Aspartic Acid/chemical synthesis , Caspase Inhibitors , Enzyme Inhibitors/chemical synthesis , Salicylates/chemical synthesis , Sulfonamides/chemical synthesis , Aspartic Acid/analogs & derivatives , Aspartic Acid/chemistry , Benzoxazoles/chemical synthesis , Benzoxazoles/chemistry , Binding Sites , Caspase 3 , Caspases/chemistry , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Fluorenes/chemical synthesis , Fluorenes/chemistry , Humans , Protein Binding , Salicylates/chemistry , Sulfonamides/chemistry , Thiophenes/chemical synthesis , Thiophenes/chemistry
18.
J Org Chem ; 64(18): 6528-6529, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-11674653
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