AVERON notebook to discover actionable cancer vulnerabilities enabled by neomorph protein-protein interactions.

Genomic alterations, such as missense mutations, often lead to the activation of oncogenic pathways and cell transformation by rewiring protein-protein interaction (PPI) networks. Understanding how mutant-directed neomorph PPIs (neoPPIs) drive cancer is vital to developing new personalized clinical strategies. However, the experimental interrogation of neoPPI functions in patients with cancer is highly challenging. To address this challenge, we developed a computational platform, termed AVERON for discovering actionable vulnerabilities enabled by rewired oncogenic networks. AVERON enables rapid systematic profiling of the clinical significance of neomorph PPIs across different cancer types, informing molecular mechanisms of neoPPI-driven tumorigenesis, and revealing therapeutically actionable neoPPI-regulated genes. We demonstrated the application of the AVERON platform by evaluating the biological functions and clinical significance of 130 neomorph interactions, experimentally determined for oncogenic BRAFV600E. The AVERON application to broad sets of mutant-directed PPIs may inform new testable biological models and clinical strategies in cancer.

Pharmacological suppression of the OTUD4/CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers.

Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-ß signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.

Antiviral response and HIV-1 inhibition in sickle cell disease.

Sickle cell disease (SCD) is characterized by hemolysis, vaso-occlusion, and ischemia. HIV-1 infection was previously shown to be suppressed in SCD PBMCs. Here, we report that HIV-1 suppression is attributed to the increased expression of iron, hypoxia, and interferon-induced innate antiviral factors. Inhibition of upregulated antiviral genes, HMOX-1, CDKN1A, and CH25H, increased HIV-1 replication in SCD PBMCs, suggesting their critical role in HIV-1 suppression. Levels of IFN-ß were elevated in SCD patients. Sickle cell hemoglobin (HbS) treatment of THP-1-derived and primary monocyte-derived macrophages induced production of IFN-ß, upregulated antiviral gene expression, and suppressed HIV-1 infection. Infection with mouse-adapted EcoHIV was suppressed in the SCD mice that also exhibited elevated levels of antiviral restriction factors. Our findings suggest that hemolysis and release of HbS leads to the induction of IFN-ß production, induction of cellular antiviral state by the expression of iron and IFN-driven factors, and suppression of HIV-1 infection.

Synthesis of Tetrazines from N-Allenylpyrrole-2-carbaldehydes and pH-Controlled Reversible Fragmentation.

A one-pot highly selective approach to the synthesis of hitherto unknown tetrahydropyrrolo[2',1':3,4]pyrazino[1,2-b]pyrrolo[2',1':3,4]pyrazino[1,2-e][1,2,4,5]tetrazine ensembles from simple and available N-allenylpyrrole-2-carbaldehydes and hydrazines has been developed. The reaction proceeds in a very facile manner and tolerates different substituents in both pyrroles and hydrazines. The novel class of organic compounds, tetrahydrodipyrrolodipyrazinotetrazines, proves to be promising pH-sensitive switchers to deliver N-aminopyrrolopyrazinium salts in acidic media and then again tetrahydrodipyrrolodipyrazinotetrazines in basic media. Both transformations give the products in quantitative yields.

SARS-CoV-2 Impact on Red Blood Cell Morphology.

Severe COVID-19 alters the biochemical and morphological characteristics of blood cells in a wide variety of ways. To date, however, the vast majority of research has been devoted to the study of leukocytes, while erythrocyte morphological changes have received significantly less attention. The aim of this research was to identify erythrocyte morphology abnormalities that occur in COVID-19, compare the number of different poikilocyte types, and measure erythrocyte sizes to provide data on size dispersion. Red blood cells obtained from 6 control donors (800-2200 cells per donor) and 5 COVID-19 patients (800-1900 cells per patient) were examined using low-voltage scanning electron microscopy. We did not discover any forms of erythrocyte morphology abnormalities that would be specific to COVID-19. Among COVID-19 patients, we observed an increase in the number of acanthocytes (p = 0.01) and a decrease in the number of spherocytes (p = 0.03). In addition, our research demonstrates that COVID-19 causes an increase in the median (p = 0.004) and interquartile range (p = 0.009) when assessing erythrocyte size. The limitation of our study is a small number of participants.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Evaluation of software impact designed for biomedical research: Are we measuring what's meaningful?

Software is vital for the advancement of biology and medicine. Through analysis of usage and impact metrics of software, developers can help determine user and community engagement. These metrics can be used to justify additional funding, encourage additional use, and identify unanticipated use cases. Such analyses can help define improvement areas and assist with managing project resources. However, there are challenges associated with assessing usage and impact, many of which vary widely depending on the type of software being evaluated. These challenges involve issues of distorted, exaggerated, understated, or misleading metrics, as well as ethical and security concerns. More attention to the nuances, challenges, and considerations involved in capturing impact across the diverse spectrum of biological software is needed. Furthermore, some tools may be especially beneficial to a small audience, yet may not have comparatively compelling metrics of high usage. Although some principles are generally applicable, there is not a single perfect metric or approach to effectively evaluate a software tool's impact, as this depends on aspects unique to each tool, how it is used, and how one wishes to evaluate engagement. We propose more broadly applicable guidelines (such as infrastructure that supports the usage of software and the collection of metrics about usage), as well as strategies for various types of software and resources. We also highlight outstanding issues in the field regarding how communities measure or evaluate software impact. To gain a deeper understanding of the issues hindering software evaluations, as well as to determine what appears to be helpful, we performed a survey of participants involved with scientific software projects for the Informatics Technology for Cancer Research (ITCR) program funded by the National Cancer Institute (NCI). We also investigated software among this scientific community and others to assess how often infrastructure that supports such evaluations is implemented and how this impacts rates of papers describing usage of the software. We find that although developers recognize the utility of analyzing data related to the impact or usage of their software, they struggle to find the time or funding to support such analyses. We also find that infrastructure such as social media presence, more in-depth documentation, the presence of software health metrics, and clear information on how to contact developers seem to be associated with increased usage rates. Our findings can help scientific software developers make the most out of the evaluations of their software so that they can more fully benefit from such assessments.

Ebola Virus NP Binding to Host Protein Phosphatase-1 Regulates Capsid Formation.

The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.

Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer's disease.

Recent genome-wide association studies have revealed genetic risk factors for Alzheimer's disease (AD) that are exclusively expressed in microglia within the brain. A proteomics approach identified moesin (MSN), a FERM (four-point-one ezrin radixin moesin) domain protein, and the receptor CD44 as hub proteins found within a co-expression module strongly linked to AD clinical and pathological traits as well as microglia. The FERM domain of MSN interacts with the phospholipid PIP2 and the cytoplasmic tails of receptors such as CD44. This study explored the feasibility of developing protein-protein interaction inhibitors that target the MSN-CD44 interaction. Structural and mutational analyses revealed that the FERM domain of MSN binds to CD44 by incorporating a beta strand within the F3 lobe. Phage-display studies identified an allosteric site located close to the PIP2 binding site in the FERM domain that affects CD44 binding within the F3 lobe. These findings support a model in which PIP2 binding to the FERM domain stimulates receptor tail binding through an allosteric mechanism that causes the F3 lobe to adopt an open conformation permissive for binding. High-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction, and one compound series was further optimized for biochemical activity, specificity, and solubility. The results suggest that the FERM domain holds potential as a drug development target. The small molecule preliminary leads generated from the study could serve as a foundation for additional medicinal chemistry effort with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Biologically inspired robotic perception-action for soft fruit harvesting in vertical growing environments.

Multiple interlinked factors like demographics, migration patterns, and economics are presently leading to the critical shortage of labour available for low-skilled, physically demanding tasks like soft fruit harvesting. This paper presents a biomimetic robotic solution covering the full 'Perception-Action' loop targeting harvesting of strawberries in a state-of-the-art vertical growing environment. The novelty emerges from both dealing with crop/environment variance as well as configuring the robot action system to deal with a range of runtime task constraints. Unlike the commonly used deep neural networks, the proposed perception system uses conditional Generative Adversarial Networks to identify the ripe fruit using synthetic data. The network can effectively train the synthetic data using the image-to-image translation concept, thereby avoiding the tedious work of collecting and labelling the real dataset. Once the harvest-ready fruit is localised using point cloud data generated by a stereo camera, our platform's action system can coordinate the arm to reach/cut the stem using the Passive Motion Paradigm framework inspired by studies on neural control of movement in the brain. Results from field trials for strawberry detection, reaching/cutting the stem of the fruit, and extension to analysing complex canopy structures/bimanual coordination (searching/picking) are presented. While this article focuses on strawberry harvesting, ongoing research towards adaptation of the architecture to other crops such as tomatoes and sweet peppers is briefly described. Supplementary Information: The online version contains supplementary material available at 10.1007/s11119-023-10000-4.

Secretory Phospholipase A2 and Interleukin-6 Levels as Predictive Markers of the Severity and Outcome of Patients with COVID-19 Infections.

Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation.

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1-infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1-infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-ß, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (>20-fold, p = 1.37 × 10-9), followed by the reduced phosphorylation of transforming growth factor-beta 2 at Ser-46 (TGF-ß2, >12-fold, p = 1.37 × 10-3). Downregulation of NPM1's Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1-Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-ß pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.

Flash drug release from nanoparticles accumulated in the targeted blood vessels facilitates the tumour treatment.

Tumour microenvironment hinders nanoparticle transport deep into the tissue precluding thorough treatment of solid tumours and metastatic nodes. We introduce an anticancer drug delivery concept termed FlaRE (Flash Release in Endothelium), which represents alternative to the existing approaches based on enhanced permeability and retention effect. This approach relies on enhanced drug-loaded nanocarrier accumulation in vessels of the target tumour or metastasised organ, followed by a rapid release of encapsulated drug within tens of minutes. It leads to a gradient-driven permeation of the drug to the target tissue. This pharmaceutical delivery approach is predicted by theoretical modelling and validated experimentally using rationally designed MIL-101(Fe) metal-organic frameworks. Doxorubicin-loaded MIL-101 nanoparticles get swiftly trapped in the vasculature of the metastasised lungs, disassemble in the blood vessels within 15 minutes and release drug, which rapidly impregnates the organ. A significant improvement of the therapeutic outcome is demonstrated in animal models of early and late-stage B16-F1 melanoma metastases with 11-fold and 4.3-fold decrease of pulmonary melanoma nodes, respectively.

Label-Free Detection of the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein at Physiologically Relevant Concentrations Using Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman scattering (SERS) spectroscopy is a surface- or cavity-enhanced variant of Raman scattering spectroscopy that allows the detection of analytes with a sensitivity down to single molecules. This method involves the use of SERS-active surfaces or cavities capable of concentrating incident radiation into small mode volumes containing the analyte. Here, we have engineered an ultranarrow metal-dielectric nano-cavity out of a film of the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) glycoprotein and a silver surface, held together by interaction between reduced protein sulfhydryl groups and silver. The concentration of light in this nano-cavity allows the label-free recording of the characteristic Raman spectra of protein samples smaller than 1 pg. This is sufficient for the ultrasensitive detection of viral protein antigens at physiologically relevant levels. Moreover, the protein SERS signal can be increased by several orders of magnitude by coating the RBD film with a nanometer-thick silver shell, thereby raising the cavity Q-factor. This ensures a sub-femtogram sensitivity of the viral antigen detection. A simple theoretical model explaining the observed additional enhancement of the SERS signal from the silver-coated protein is proposed. Our study is the first to obtain the characteristic Raman and SERS spectra of the RBD of S glycoprotein, the key SARS-CoV-2 viral antigen, directly, without the use of Raman-reporter molecules. Thus, our approach allows label-free recording of the characteristic spectra of viral antigens at concentrations orders of magnitude lower than those required for detecting the whole virus in biological media. This makes it possible to develop a high-performance optical detection method and conformational analysis of the pathogen and its variants.

Prevalence and Dynamics of SARS-CoV-2 Antibodies in the Population of St. Petersburg, Russia.

BACKGROUND: The aim of the study was to assess the prevalence of seropositive status for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-IgA, -IgM, and -IgG; its dynamics in connection with restrictive measures during the coronavirus disease (COVID-19) pandemic; and the quantitative dynamics of antibody levels in the population of St. Petersburg, Russia. METHODS: From May to November 2020, a retrospective analysis of Saint Petersburg State University Hospital laboratory database was performed. The database included 158,283 test results of 87,067 patients for SARS-CoV-2 detection by polymerase chain reaction (PCR) and antibody detection of SARS-CoV-2-IgA, -IgM, and -IgG. The dynamics of antibody level was assessed using R v.3.6.3. RESULTS: The introduction of a universal lockdown was effective in containing the spread of COVID-19. The proportion of seropositive patients gradually decreased; approximately 50% of these patients remained seropositive for IgM after 3-4 weeks; for IgG, by follow-up week 22; and for IgA, by week 12. The maximum decrease in IgG and IgA was observed 3-4 months and 2 months after the detection of the seropositive status, respectively. CONCLUSIONS: The epidemiological study of post-infection immunity to COVID-19 demonstrates significant differences in the dynamics of IgA, IgM, and IgG seropositivity and in PCR test results over time, which is linked to the introduction of restrictive measures. Both the proportion of seropositive patients and the level of all antibodies decreased in terms of the dynamics, and only approximately half of these patients remained IgG-positive 6 months post-infection.

Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Solvent Moisture-Controlled Self-Assembly of Fused Benzoimidazopyrrolopyrazines with Different Ring's Interposition.

This article shows that two extremely important families of fused heterocyclic assemblies, namely 6-methylbenzo[4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine and 5a-methyl-5a,6-dihydro-5H,12H-benzo[4,5]imidazo[1,2-a]pyrrolo[1,2-d]pyrazine, can be synthesized from only two available building blocks (N-allenylpyrrole-2-carbaldehyde and o-phenylenediamine) by controlling only one reaction parameter (water content of the medium). It should be emphasized that the latter class of compounds (with an a/d arrangement) is previously unknown. If the allene group is introduced not into the starting compound, but during the reaction (in superbase media), a heterocyclic ensemble, 5-methylbenzo[4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazines, with a different position of the methyl group is formed.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain.

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 µM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e- ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.