ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.
Subject(s)
Nebulizers and Vaporizers , Pulmonary Disease, Chronic Obstructive , Humans , Adrenergic beta-2 Receptor Agonists/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Administration, Inhalation , Bronchodilator Agents/therapeutic use , Drug Combinations , Muscarinic Antagonists/therapeutic use , Drug Therapy, CombinationABSTRACT
ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.
Subject(s)
COVID-19 , Peptide Hydrolases , Antiviral Agents , Binding Sites , Humans , Ligands , Protease Inhibitors , RNA, Viral , SARS-CoV-2ABSTRACT
Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.
Subject(s)
Carcinogenesis/pathology , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphoprotein Phosphatases/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Phosphoprotein Phosphatases/genetics , Phosphorylation , RNA, Small Interfering/metabolism , Substrate SpecificityABSTRACT
Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Methicillin-Resistant Staphylococcus aureus/physiology , Staphylococcal Infections/immunology , Staphylococcal Toxoid/immunology , Staphylococcal Vaccines/immunology , Animals , Antibodies, Bacterial/blood , Antibody Formation , Broadly Neutralizing Antibodies/blood , Immunity, Heterologous , Immunogenicity, Vaccine , Lymphocyte Activation , Macaca mulatta , Superantigens/immunology , VaccinationABSTRACT
BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) are more likely to be readmitted than patients with other chronic medical conditions, yet knowledge regarding such readmissions is limited. We aimed to determine factors associated with readmission within 30 days of a COPD hospitalization or death with an emphasis on examining aspects of socioeconomic status and specific comorbidities. METHODS: A population-based cohort study was conducted using health administrative data from Ontario, Canada. All hospitalizations for COPD between 2004 and 2014 were considered. The primary exposures were socioeconomic status as measured by residential instability (an ecologic variable), and comorbidities such as cardiovascular disease and cancer. Other domains of socioeconomic status were considered as secondary exposures. Logistic regression with generalized estimating equations was used to examine the effect of exposures, adjusting for other patient factors, on 30-day readmission or death. RESULTS: There were 126,013 patients contributing to 252,756 index COPD hospitalizations from 168 Ontario hospitals. Of these hospitalizations, 19.4% resulted in a readmission and 2.8% resulted in death within 30 days. After adjusting for other factors, readmissions or death were modestly more likely among people with the highest residential instability compared to the lowest (OR 1.05, 95% CI 1.01-1.09). Comorbidities such as cardiovascular disease and cancer, as well as other aspects of low socioeconomic status also increased readmission or death risk. INTERPRETATION: Socioeconomic status, measured in various ways, and many comorbidities predict 30-day readmission or death in patients hospitalized for COPD. Strategies that address these factors may help reduce readmissions and death.
Subject(s)
Patient Readmission , Pulmonary Disease, Chronic Obstructive/epidemiology , Adult , Aged , Aged, 80 and over , Cohort Studies , Comorbidity , Female , Humans , Logistic Models , Male , Middle Aged , Ontario/epidemiology , Patient Readmission/statistics & numerical data , Pulmonary Disease, Chronic Obstructive/mortality , Retrospective Studies , Risk Factors , Social Class , Time FactorsABSTRACT
BACKGROUND: There is limited knowledge on what proportions of patients with COPD receive ambulatory care from primary care physicians, pulmonologists, or other specialists. We evaluated the types and combinations of physicians who provide ambulatory care to patients with COPD. METHODS: We conducted a population-based cross-sectional study using health administrative datasets from Ontario, Canada between April 1, 2014 and March 31, 2015. Individuals age 35 years and older with physician-diagnosed COPD were identified, using a previously validated COPD case definition. The primary outcomes were ambulatory visits to primary care physicians, pulmonologists, and all other specialists within a 1-year period. RESULTS: There were 895,155 individuals identified as having physician-diagnosed COPD. Of those, 56,533 individuals (6.3%) had no ambulatory care visits, 802,327 (89.6%) saw primary care physicians, and 95,782 (10.7%) consulted pulmonologists. By comparison, 736,496 (82.3%) saw other specialists, and 218,997 (24.5%) saw cardiologists. There were 32,473 individuals (3.6%) who underwent COPD-related hospitalizations. Of those in the subcohort with one hospitalization, about 30.0% saw pulmonologists; 43.7% of those who underwent two or more hospitalizations saw pulmonologists, and 9.9% with no hospitalization consulted pulmonologists. CONCLUSIONS: Primary care physicians play a substantial role in caring for patients with COPD. But only one-half as many patients with COPD saw pulmonologists than cardiologists, suggesting that COPD may receive less specialty care compared with other chronic medical conditions. This information can help inform COPD care strategies to improve COPD care and minimize exacerbations and associated health-care costs. It also suggests a need for more research to provide guidance on when patients with COPD should be referred to pulmonologists.
Subject(s)
Ambulatory Care/methods , Physician's Role , Physicians, Primary Care/statistics & numerical data , Pulmonary Disease, Chronic Obstructive/therapy , Pulmonologists/statistics & numerical data , Adult , Aged , Cross-Sectional Studies , Female , Humans , Male , Middle Aged , Morbidity/trends , Ontario/epidemiology , Pulmonary Disease, Chronic Obstructive/epidemiology , Retrospective StudiesABSTRACT
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.
Subject(s)
Antigens, Bacterial/pharmacology , Apoptosis Regulatory Proteins/metabolism , Bacillus anthracis/chemistry , Bacterial Toxins/pharmacology , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Amino Acid Substitution , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Apoptosis Regulatory Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Cell Line, Tumor , MAP Kinase Signaling System/genetics , Mice , Mitogen-Activated Protein Kinase Kinases/genetics , Mutation, Missense , PhosphorylationABSTRACT
To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function.
Subject(s)
Chromones/pharmacology , Guaiacol/analogs & derivatives , Leukemia, Myeloid, Acute/metabolism , Lysosomes/drug effects , Tubulin Modulators/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Guaiacol/pharmacology , Humans , Leukemia, Myeloid, Acute/pathology , Lysosomes/chemistry , Lysosomes/metabolism , Mice , Saccharomyces cerevisiae , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
Reversible and localized blood-brain barrier disruption (BBBD) using focused ultrasound (FUS) in combination with intravascularly administered microbubbles (MBs) has been established as a non-invasive method for drug delivery to the brain. Using two-photon fluorescence microscopy (2 PFM), we imaged the cerebral vasculature during BBBD and observed the extravasation of fluorescent dye in real-time in vivo. We measured the enhanced permeability upon BBBD for both 10 kDa and 70 kDa dextran conjugated Texas Red (TR) at the acoustic pressure range of 0.2-0.8 MPa and found that permeability constants of TR10 kDa and TR70 kDa vary from 0.0006 to 0.0359 min(-1) and from 0.0003 to 0.0231 min(-1), respectively. For both substances, a linear regression was applied on the permeability constant against the acoustic pressure and the slope from best-fit was found to be 0.039 ± 0.005 min(-1)/MPa and 0.018 ± 0.005 min(-1)/MPa, respectively. In addition, the pressure threshold for successfully induced BBBD was confirmed to be 0.4-0.6MPa. Finally, we identified two types of leakage kinetics (fast and slow) that exhibit distinct permeability constants and temporal disruption onsets, as well as demonstrated their correlations with the applied acoustic pressure and vessel diameter. Direct assessment of vascular permeability and insights on its dependency on acoustic pressure, vessel size and leakage kinetics are important for treatment strategies of BBBD-based drug delivery.
Subject(s)
Blood-Brain Barrier/metabolism , Brain/blood supply , Dextrans/administration & dosage , Drug Delivery Systems/instrumentation , Fluorescent Dyes/administration & dosage , Ultrasonics/instrumentation , Xanthenes/administration & dosage , Animals , Capillary Permeability , Equipment Design , Male , Microscopy, Fluorescence, Multiphoton , Rats , Rats, WistarABSTRACT
Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.
Subject(s)
Intracellular Membranes/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Lysosomes/metabolism , Neoplastic Stem Cells/metabolism , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Cell Survival/drug effects , Female , Gene Knockdown Techniques , Genome-Wide Association Study , Humans , Intracellular Membranes/pathology , K562 Cells , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Lysosomal-Associated Membrane Protein 2 , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Lysosomes/genetics , Lysosomes/physiology , Male , Mefloquine/pharmacokinetics , Mefloquine/pharmacology , Mice , Neoplastic Stem Cells/pathology , Permeability/drug effects , Saccharomyces cerevisiae/geneticsABSTRACT
Blood-brain barrier (BBB) disruption can be achieved with ultrasound (US) and circulating microbubble (MB) contrast agent. Using dorsal US sonication and Definity, an MB contrast agent, responses of the cortical cerebral vasculature to BBB opening were observed with varying acoustic peak negative pressure (0.071 to 0.25 MPa) under two-photon microscope. Wistar rats with a craniotomy were sonicated with a single piezoelectric transducer following the intravenous injection of Texas Red for visualization of vasculature and leakage from BBB opening. Based on time-dependent intensity change in the extravascular area, the leakage was classified into three types: fast, sustained, and slow. Fast leakage was characterized by a rapid increase to peak intensity during sonication, but a decrease afterwards, occurring at all pressures and vessels sizes analyzed in our study. Sustained leakage was indicated by a similar, immediate increase to peak intensity but one that remained elevated for the duration of imaging, occurring at low-to-intermediate pressures. Slow leakage began 5 to 15 minutes after sonication, dominating at low pressures, and was more prevalent among smaller vessels than fast and sustained leakage. Our study showed the possibility of controlling leakage type and vessel size in US-induced BBB opening through varying acoustic pressure.