scholarly journals A Strategy for Combinatorial Cavity Design in De Novo Proteins

Life ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 9 ◽  
Author(s):  
Christina Karas ◽  
Michael Hecht
Keyword(s):  
De Novo ◽  
Protein S ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Functional Sites ◽  
Helical Proteins ◽  
Living Organisms ◽  
Protein Sequence Space

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Current Challenges in the Development of Vaccines and Drugs Against Emerging Vector-borne Diseases

2019 ◽  
Vol 26 (16) ◽  
pp. 2974-2986 ◽  
Author(s):  
Kwang-sun Kim
Keyword(s):  
New Host ◽  
In Vivo Models ◽  
Vector Borne Diseases ◽  
Novel Drugs ◽  
Huge Impact ◽  
Tick Borne Disease ◽  
Vector Borne ◽  
Living Organisms

Vectors are living organisms that transmit infectious diseases from an infected animal to humans or another animal. Biological vectors such as mosquitoes, ticks, and sand flies carry pathogens that multiply within their bodies prior to delivery to a new host. The increased prevalence of Vector-Borne Diseases (VBDs) such as Aedes-borne dengue, Chikungunya (CHIKV), Zika (ZIKV), malaria, Tick-Borne Disease (TBD), and scrub typhus has a huge impact on the health of both humans and livestock worldwide. In particular, zoonotic diseases transmitted by mosquitoes and ticks place a considerable burden on public health. Vaccines, drugs, and vector control methods have been developed to prevent and treat VBDs and have prevented millions of deaths. However, development of such strategies is falling behind the rapid emergence of VBDs. Therefore, a comprehensive approach to fighting VBDs must be considered immediately. In this review, I focus on the challenges posed by emerging outbreaks of VBDs and discuss available drugs and vaccines designed to overcome this burden. Research into promising drugs needs to be upgraded and fast-tracked, and novel drugs or vaccines being tested in in vitro and in vivo models need to be moved into human clinical trials. Active preventive tactics, as well as new and upgraded diagnostics, surveillance, treatments, and vaccination strategies, need to be monitored constantly if we are to manage VBDs of medical importance.

scholarly journals An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marisa Nacke ◽  
Emma Sandilands ◽  
Konstantina Nikolatou ◽  
Álvaro Román-Fernández ◽  
Susan Mason ◽  
...  
Keyword(s):  
Metastatic Cancer ◽  
Cellular Responses ◽  
Signalling Pathways ◽  
External Signal ◽  
Phosphoinositide Metabolism ◽  
Invasion And Metastasis ◽  
3 Dimensional

AbstractThe signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

Computational study for suppression of CD25/IL-2 interaction

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Moein Dehbashi ◽  
Zohreh Hojati ◽  
Majid Motovali-bashi ◽  
Mazdak Ganjalikhani-Hakemi ◽  
Akihiro Shimosaka ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cancer Progression ◽  
Immune Escape ◽  
De Novo ◽  
Computational Study ◽  
Treg Cells ◽  
Homology Search ◽  
Small Interfering Rnas

AbstractCancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

scholarly journals EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii98-ii98
Author(s):  
Anne Marie Barrette ◽  
Alexandros Bouras ◽  
German Nudelman ◽  
Zarmeen Mussa ◽  
Elena Zaslavsky ◽  
...  
Keyword(s):  
Survival Benefit ◽  
De Novo ◽  
Epithelial Mesenchymal Transition ◽  
Intraperitoneal Administration ◽  
Standard Of Care ◽  
Tumor Burden ◽  
Mesenchymal Transition ◽  
Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

scholarly journals The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma

2020 ◽  
Vol 21 (15) ◽  
pp. 5499
Author(s):  
Hannah L. Smith ◽  
Stephen A. Beers ◽  
Juliet C. Gray ◽  
Janos M. Kanczler
Keyword(s):  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
3D Models ◽  
In Ovo ◽  
Future Directions ◽  
3 Dimensional ◽  
In Vivo Animal Models

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

scholarly journals PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Nan Huang ◽  
Chang Xu ◽  
Liang Deng ◽  
Xue Li ◽  
Zhixuan Bian ◽  
...  
Keyword(s):  
Dna Damage ◽  
Histone Deacetylase ◽  
Dna Damage Response ◽  
De Novo ◽  
Dna Damage Repair ◽  
Histone Deacetylase 1 ◽  
Damage Repair ◽  
Damage Response

AbstractPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.

Hippokampale Estrogensynthese und synaptische Plastizität

e-Neuroforum ◽  
2007 ◽  
Vol 13 (4) ◽  
Author(s):  
Lars Fester ◽  
Janine Prange-Kiel ◽  
Gabriele M. Rune
Keyword(s):  
De Novo ◽  
Synaptische Plastizität

ZusammenfassungUnsere Untersuchungen der letzten Jahre haben gezeigt, dass nicht das Ovar die Quelle für Estrogen induzierte synaptische Plastizität im Hippokampus ist, sondern dieses aus dem Hippokampus selber stammt und haben damit einen Paradigmawechsel eingeleitet, der Estrogen als Neuromodulator unabhängig vom Geschlecht identifiziert. Hippokampale Neurone von Ratten beiderlei Geschlechts sind in der Lage, aus Cholesterol Estrogene de novo zu synthetisieren. Diese hippokampale Estrogensynthese ist sowohl für den Erhalt von Spinesynapsen in vivo als auch in vitro essenziell. Die Hemmung der Estrogensynthese zieht einen Synapsenverlust nach sich und Langzeitpotenzierung ist nicht mehr induzierbar. Die Effekte von hippokampalem Estrogen sind auto-/parakriner Natur, die über die bekannten Estrogenrezeptor-Subtypen, ERα und ERβ, vermittelt werden. Die Regulation der hippokampalen Estrogensynthese erfolgt über GnRH und erklärt die Korrelation der Spinesynapsendichte mit dem weiblichen genitalen Zyklus, die für den Hippokampus spezifisch ist.

scholarly journals Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mylène Tajan ◽  
Marc Hennequart ◽  
Eric C. Cheung ◽  
Fabio Zani ◽  
Andreas K. Hock ◽  
...  
Keyword(s):  
Therapeutic Efficacy ◽  
De Novo ◽  
Carbon Availability ◽  
Global Protein Synthesis ◽  
Enhanced Expression ◽  
One Carbon Metabolism ◽  
Amino Acid Depletion ◽  
Pathway Inhibition

AbstractMany tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.

Lipidated Methotrexate Microbubbles: A Promising Rheumatoid Arthritis Theranostic Medicine Manipulated via Ultrasonic Irradiation

2021 ◽  
Vol 17 (7) ◽  
pp. 1293-1304
Author(s):  
Zhuofei Zhao ◽  
Xiaona Lin ◽  
Lulu Zhang ◽  
Xia Liu ◽  
Qingwen Wang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
De Novo ◽  
Low Ph ◽  
Curative Effect ◽  
Inflammatory Microenvironment ◽  
Raw 264.7 Cell ◽  
Infection Focus ◽  
Raw 264.7 Cell Line

De novo designed lipidated methotrexate was synthesized and self-assembled into microbubbles for targeted rheumatoid arthritis theranostic treatment. Controlled lipidatedmethotrexate delivery was achieved by ultrasound-targetedmicrobubble destruction technique. Methotrexate was dissociated inflammatory microenvironment of synovial cavity, owing to representive low pH and enriched leucocyte esterase. We first manipulated methotrexate controlled release with RAW 264.7 cell line in vitro and further verified with rheumatoid arthritis rabbits in vivo. Results showed that lipidated methotrexate microbubbles precisely affected infection focus and significantly enhanced rheumatoid arthritis curative effect comparing with dissociative methotrexate. This study indicates that lipidated methotrexate microbubbles might be considered as a promising rheumatoid arthritis theranostics medicine.

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