Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin\u27s high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 muM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE: Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors

Clean, green, and not so mean: Can business save the world?

No matter how you define it, corporate social responsibility (CSR) is a hot topic. From community investing to business ethics to environmental sustainability and beyond, proponents of CSR view the business landscape through a lens that focuses less on profitability and more on the greater good. “Doing well by doing good” is the business world’s new mantra. Concepts of CSR, green business, social entrepreneurship, and peace through commerce have become a focus of research and are inspiring the next generation of businesspeople. The 2010 BRASS Program gave attendees an expert’s view of certain niches within the broader realm of ethical or socially responsible business practices

Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease [preprint]

Hepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 Angstrom away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the α-2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site

Severe Radiation-Induced Lymphopenia During Concurrent Chemoradiotherapy for Stage III Non-Small Cell Lung Cancer: External Validation of Two Prediction Models

BACKGROUND: Severe radiation-induced lymphopenia (RIL) in patients undergoing chemoradiotherapy (CRT) for non-small cell lung cancer (NSCLC) is associated with decreased immunotherapy efficacy and survival. At The Christie and MD Anderson Cancer Center (MDACC), prediction models for lymphopenia were developed in lung and esophageal cancer patients, respectively. The aim of this study was to externally validate both models in patients with stage III NSCLC. METHODS: Patients who underwent concurrent CRT for stage III NSCLC in 2019-2021 were studied. Outcomes were grade ≥3 and grade 4 lymphopenia during CRT. The Christie model predictors for grade ≥3 lymphopenia included age, baseline lymphocyte count, radiotherapy duration, chemotherapy, mean heart and lung doses, and thoracic vertebrae V20Gy. MDACC predictors for grade 4 lymphopenia were age, baseline lymphocyte count, planning target volume (PTV), and BMI. The external performance of both models was assessed. RESULTS: Among 100 patients, 78 patients (78%) developed grade ≥3 lymphopenia, with grade 4 lymphopenia in 17 (17%). For predicting grade ≥3 lymphopenia, the Christie and MDACC models yielded CONCLUSION: The PTV-based MDACC prediction model for severe RIL demonstrated superior external performance in NSCLC patients compared to the dosimetry-based Christie model. As such, the MDACC model can aid in identifying patients at high risk for severe lymphopenia. However, to optimize radiotherapy planning, further improvement and external validation of dosimetry-based models is desired

Prognostic and predictive markers of systemic sclerosis-interstitial lung disease in a clinical trial and long-term observational cohort

OBJECTIVES: Explore prognostic and predictive markers of systemic sclerosis-associated interstitial lung disease (SSc-ILD) outcomes in a phase 3 trial (focuSSced) and prognostic markers in a real-world cohort (SMART). METHODS: The focuSSced SSc-ILD subgroup included 68 of 106 placebo-treated and 68 of 104 tocilizumab-treated patients. The SMART cohort included 505 patients with SSc-ILD. Linear mixed-effect models were used to identify factors associated with change in forced vital capacity (FVC). Kaplan-Meier estimation and Cox regression were used for time-to-event analyses. RESULTS: In placebo-treated focuSSced patients, sex was a significant prognostic factor for FVC decline; males had increased risk for absolute decline ≥10% in percent-predicted FVC (ppFVC) and 0.22% faster weekly FVC decline than females (P = 0.0001). FVC was 9.8% lower in patients with C-reactive protein >6 mg/ml versus those with C-reactive protein ≤6 mg/ml (P = 0.0059). Tocilizumab reduced the risk for ≥10% decline in ppFVC in patients who were male, had earlier disease (<2 years duration), had interleukin-6 levels <10 pg/ml, or had anti-topoisomerase antibodies (ATA). In the SMART cohort, prognostic factors for ppFVC <70% were male sex, ATA, and low baseline FVC. Males had 3.3% lower FVC 1 year after disease onset (P < 0.001) and 0.6% faster yearly decline (P = 0.03) than females. CONCLUSION: Prognostic markers in SSc-ILD were similar between focuSSced and SMART. Male sex and inflammatory markers were associated with lower FVC but interleukin-6 ≥ 10 pg/ml was not predictive of response to tocilizumab. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02453256

Genetic Pharmacotherapy as an Early CNS Drug Development Strategy: Testing Glutaminase Inhibition for Schizophrenia Treatment in Adult Mice

Genetic pharmacotherapy is an early drug development strategy for the identification of novel CNS targets in mouse models prior to the development of specific ligands. Here for the first time, we have implemented this strategy to address the potential therapeutic value of a glutamate-based pharmacotherapy for schizophrenia involving inhibition of the glutamate recycling enzyme phosphate-activated glutaminase. Mice constitutively heterozygous for GLS1, the gene encoding glutaminase, manifest a schizophrenia resilience phenotype, a key dimension of which is an attenuated locomotor response to propsychotic amphetamine challenge. If resilience is due to glutaminase deficiency in adulthood, then glutaminase inhibitors should have therapeutic potential. However, this has been difficult to test given the dearth of neuroactive glutaminase inhibitors. So, we used genetic pharmacotherapy to ask whether adult induction of GLS1 heterozygosity would attenuate amphetamine responsiveness. We generated conditional floxGLS1 mice and crossed them with global CAGERT2cre∕+ mice to produce GLS1 iHET mice, susceptible to tamoxifen induction of GLS1 heterozygosity. One month after tamoxifen treatment of adult GLS1 iHET mice, we found a 50% reduction in GLS1 allelic abundance and glutaminase mRNA levels in the brain. While GLS1 iHET mice showed some recombination prior to tamoxifen, there was no impact on mRNA levels. We then asked whether induction of GLS heterozygosity would attenuate the locomotor response to propsychotic amphetamine challenge. Before tamoxifen, control and GLS1 iHET mice did not differ in their response to amphetamine. One month after tamoxifen treatment, amphetamine-induced hyperlocomotion was blocked in GLS1 iHET mice. The block was largely maintained after 5 months. Thus, a genetically induced glutaminase reduction—mimicking pharmacological inhibition—strongly attenuated the response to a propsychotic challenge, suggesting that glutaminase may be a novel target for the pharmacotherapy of schizophrenia. These results demonstrate how genetic pharmacotherapy can be implemented to test a CNS target in advance of the development of specific neuroactive inhibitors. We discuss further the advantages, limitations, and feasibility of the wider application of genetic pharmacotherapy for neuropsychiatric drug development

Prostate Cancer-Induced Endothelial-Cell-to-Osteoblast Transition Drives Immunosuppression in the Bone-Tumor Microenvironment Through Wnt Pathway-Induced M2 Macrophage Polarization

Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells\u27 proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC

Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control.

The Hippo tumor suppressor pathway is essential for development and tissue growth control, encompassing a core cassette consisting of the Hippo (MST1/2), Warts (LATS1/2), and Tricornered (NDR1/2) kinases together with MOB1 as an important signaling adaptor. However, it remains unclear which regulatory interactions between MOB1 and the different Hippo core kinases coordinate development, tissue growth, and tumor suppression. Here, we report the crystal structure of the MOB1/NDR2 complex and define key MOB1 residues mediating MOB1's differential binding to Hippo core kinases, thereby establishing MOB1 variants with selective loss-of-interaction. By studying these variants in human cancer cells and Drosophila, we uncovered that MOB1/Warts binding is essential for tumor suppression, tissue growth control, and development, while stable MOB1/Hippo binding is dispensable and MOB1/Trc binding alone is insufficient. Collectively, we decrypt molecularly, cell biologically, and genetically the importance of the diverse interactions of Hippo core kinases with the pivotal MOB1 signal transducer.The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression

Toward 68Ga and 64Cu Positron Emission Tomography Probes: Is H2dedpa-N,N′-pram the Missing Link for dedpa Conjugation?

[Abstract] H2dedpa-N,N′-pram (H2L1), a new chelator derived from the hexadentate ligand 1,2-bis[[(6-carboxypyridin-2-yl)methyl]amino]ethane (H2dedpa), which incorporates 3-propylamine chains anchored to the secondary amines of the ethylenediamine core of the latter, has emerged as a very promising scaffold for preparing 68Ga- and 64Cu-based positron emission tomography probes. This new platform is cost-effective and easy to prepare, and the two pendant primary amines make it versatile for the preparation of bifunctional chelators by conjugation and/or click chemistry. Reported herein, we have also included the related H2dedpa-N,N′-prpta (H2L2) platform as a simple structural model for its conjugated systems. X-ray crystallography confirmed that the N4O2 coordination sphere provided by the dedpa2– core is maintained at both Ga(III) and Cu(II). The complex formation equilibria were deeply investigated by a thorough multitechnique approach with potentiometric, NMR spectrometric, and UV–vis spectrophotometric titrations, revealing effective chelation. The thermodynamic stability of the Ga(III) complexes at physiological relevant conditions is slightly higher than that of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the common and clinically approved chelator used in the clinic [pGa = 19.5 (dedpa-N,N′-pram) and 20.8 (dedpa-N,N′-prpta) versus 18.5 (DOTA) at identical conditions], and significantly higher for the Cu(II) complexes [pCu = 21.96 (dedpa-N,N′-pram) and 22.8 (dedpa-N,N′-prpta) versus 16.2 (DOTA)], which are even more stable than that of the parent ligand dedpa2– (pCu = 18.5) and that of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (pCu = 18.5). This high stability found for Cu(II) complexes is related to the conversion of the secondary amines of the ethylenediamine core of dedpa2– into tertiary amines, whereby the architecture of the new H2L1 chelator is doubly optimal in the case of this metal ion: high accessibility of the primary amine groups and their incorporation via the secondary amines, which contributes to a significant increase in the stability of the metal complex. Quantitative labeling of both chelators with both radionuclides ([68Ga]Ga3+ and [64Cu]Cu2+) was observed within 15 min at room temperature with concentrations as low as 10–5 M. Furthermore, serum stability studies confirmed a high radiochemical in vitro stability of all systems and therefore confirmed H2L1 as a promising and versatile chelator for further radiopharmaceutical in vivo studies.We thank NSERC for PGS-D and UBC for Four Year Fellowships (to L.S.), the NSERC CREATE IsoSiM training program at TRIUMF (to L.S.), as well as NSERC Discovery Grants (to C.O. and P.S.). TRIUMF receives additional funding via a contribution agreement with the National Research Council of Canada. M.d.G.J.-P. gratefully acknowledges the technical assistance of T. R. Masvikeni. C.P.-B., T.R., A.d.B., and T.R.-B. are grateful to the Spanish Ministry of Science and Innovation (CTQ2016-74862-P) and Xunta de Galicia (ED431B 2022/40) for financial support. C.P.-B. also thanks Xunta de Galicia for a Ph.D. fellowship. We also appreciate the help of Dr. A. Llamas (Unidade de Raios X RIAIDT at University of Santiago de Compostela, Spain) for useful crystallographic discussions. Funding for open access charge provided by Universidade da Coruña/CISUG.Xunta de Galicia; ED431B 2022/4