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1.
PLoS One ; 17(7): e0271066, 2022.
Article in English | MEDLINE | ID: mdl-35816490

ABSTRACT

As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection.


Subject(s)
COVID-19 Drug Treatment , Aerosols , Angiotensin-Converting Enzyme 2 , Angiotensins , Animals , Clinical Trials, Phase I as Topic , Dogs , Humans , Mice , Nebulizers and Vaporizers , Peptidyl-Dipeptidase A/metabolism , Renin/metabolism , Renin-Angiotensin System , SARS-CoV-2
2.
bioRxiv ; 2021 Sep 20.
Article in English | MEDLINE | ID: mdl-34545364

ABSTRACT

To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.

3.
Cancer Lett ; 385: 179-187, 2017 01 28.
Article in English | MEDLINE | ID: mdl-27793694

ABSTRACT

The objective of this study was to determine the therapeutic potential of polo-like kinase 1 (Plk1) inhibition in melanoma, in vivo. Employing Vectra technology, we assessed the Plk1 expression profile in benign nevi, malignant (stages I-IV) and metastatic melanomas. We found a significant elevation of Plk1 immunostaining in melanoma tissues. Further, a second generation small molecule Plk1 inhibitor, BI 6727, resulted in reductions in growth, viability and clonogenic survival, as well as an increase in apoptosis of A375 and Hs 294T melanoma cells. BI 6727 treatment also resulted in a G2/M-as well as S-phase cell cycle arrest in melanoma cells. Importantly, BI 6727 (intravenous injection; 10 and 25 mg/kg body weight) treatment resulted in significant tumor growth delay and regression in vivo in A375-and Hs 294T-implanted xenografts in athymic nude mice. These anti-melanoma effects were accompanied with a decreased cellular proliferation (Ki-67 staining) and induction of apoptosis (caspase 3 activation). In addition, BI 6727 treatment caused a marked induction of p53 and p21 in vitro as well as in vivo. Overall, we suggest that Plk1 inhibition may be a useful approach as a monotherapy as well as in combination with other existing therapeutics, for melanoma management.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Proliferation/drug effects , Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pteridines/pharmacology , Skin Neoplasms/drug therapy , Tumor Burden/drug effects , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , Ki-67 Antigen/metabolism , Melanoma/enzymology , Melanoma/secondary , Mice, Nude , Molecular Targeted Therapy , Mutation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins B-raf/genetics , S Phase Cell Cycle Checkpoints/drug effects , Signal Transduction/drug effects , Skin Neoplasms/enzymology , Skin Neoplasms/pathology , Time Factors , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays , Polo-Like Kinase 1
4.
J Invest Dermatol ; 136(10): 2041-2048, 2016 10.
Article in English | MEDLINE | ID: mdl-27251789

ABSTRACT

In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.


Subject(s)
GTP Phosphohydrolases/genetics , Melanocytes/metabolism , Melanoma/pathology , Membrane Proteins/genetics , Proteomics/methods , Skin Neoplasms/pathology , Casein Kinase II/metabolism , Humans , Mass Spectrometry/methods , Melanoma/genetics , Mitogen-Activated Protein Kinases/metabolism , Mutation , Phosphatidylinositol 3-Kinase/metabolism , Phosphopeptides/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Signal Transduction , Skin Neoplasms/genetics
5.
J Proteome Res ; 13(11): 5041-50, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-24884503

ABSTRACT

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAF(V600E) mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC-MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1's role in cancer cell survival.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Melanoma/drug therapy , Melanoma/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proteomics/methods , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Pteridines/pharmacology , Cell Cycle Proteins/metabolism , Cell Line, Tumor/drug effects , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , Humans , Melanoma/genetics , Melanoma/pathology , Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Skin Neoplasms/drug therapy , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tandem Mass Spectrometry/methods , Polo-Like Kinase 1
6.
Cancer Res ; 73(23): 6848-55, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24265276

ABSTRACT

Polo-like kinase 1 (Plk1) is a well-established mitotic regulator with a diverse range of biologic functions continually being identified throughout the cell cycle. Preclinical evidence suggests that the molecular targeting of Plk1 could be an effective therapeutic strategy in a wide range of cancers; however, that success has yet to be translated to the clinical level. The lack of clinical success has raised the question of whether there is a true oncogenic addiction to Plk1 or if its overexpression in tumors is solely an artifact of increased cellular proliferation. In this review, we address the role of Plk1 in carcinogenesis by discussing the cell cycle and DNA damage response with respect to their associations with classic oncogenic and tumor suppressor pathways that contribute to the transcriptional regulation of Plk1. A thorough examination of the available literature suggests that Plk1 activity can be dysregulated through key transformative pathways, including both p53 and pRb. On the basis of the available literature, it may be somewhat premature to draw a definitive conclusion on the role of Plk1 in carcinogenesis. However, evidence supports the notion that oncogene dependence on Plk1 is not a late occurrence in carcinogenesis and it is likely that Plk1 plays an active role in carcinogenic transformation.


Subject(s)
Carcinogenesis/genetics , Cell Cycle Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/physiology , Animals , Cell Cycle/genetics , Cell Proliferation , Genes, Tumor Suppressor/physiology , Genes, p53/physiology , Humans , Mitosis/genetics , Oncogenes/physiology , Polo-Like Kinase 1
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