Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Prognostic impact of variant histology in bladder cancer: Would early and aggressive treatment shift the paradigm?

INTRODUCTION: Bladder cancer (BC) is an increasingly frequent malignancy worldwide. Several variant histologies (VH) have been described in BC with a distinct clinical behavior. OBJECTIVES: This study aims to assess the prognostic impact of VH in BC, comparing its outcomes to pure urothelial carcinoma PUC in both non-muscle invasive (NMIBC) and muscle-invasive (MIBC) settings. METHODS: We included patients with primary BC, comparing those with VH with those with PUC, with an age and sex-matched proportion of 1:3, considering stage at diagnosis, recurrence-free, progression-free, and overall survival (OS). A total of 616 patients were included in the study, (460 UC and 151 VH). RESULTS: After first TURBT, MIBC was present in 99 (64.1%) of patients with VH, and 95 (20.6%) with UC (p<0.001). Concerning NMIBC, we observed higher rates of progression to MIBC amid patients with VH (p=0.009). Nodal involvement (p=0.020) and metastatic disease (p<0.001) were significantly higher within the VH group. A higher OS was observed among patients with NMIBC of PUC (p<0.001). There were no statistically significant differences of metastasis-free survival and OS between VH and UC groups within the MIBC setting. CONCLUSION: We confirmed that VH presents a more aggressive clinical course compared to PUC. An earlier radical treatment within the NMIBC setting could increase the oncological outcomes of the VH patients.

Deep thermal profiling for detection of functional proteoform groups.

The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.

Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.

Hepatitis E virus (HEV) causes 14 million infections and 60,000 deaths per year globally, with immunocompromised persons and pregnant women experiencing severe symptoms. Although ribavirin can be used to treat chronic hepatitis E, toxicity in pregnant patients and the emergence of resistant strains are major concerns. Therefore there is an imminent need for effective HEV antiviral agents. The aims of this study were to develop a drug screening platform and to discover novel approaches to targeting steps within the viral life cycle. We developed a screening platform for molecules inhibiting HEV replication and selected a candidate, isocotoin. Isocotoin inhibits HEV replication through interference with heat shock protein 90 (HSP90), a host factor not previously known to be involved in HEV replication. Additional work is required to understand the compound's translational potential, however this suggests that HSP90-modulating molecules, which are in clinical development as anti-cancer agents, may be promising therapies against HEV.

A computational method for detection of ligand-binding proteins from dose range thermal proteome profiles.

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. However, current data analysis strategies use preset thresholds that can lead to suboptimal sensitivity/specificity tradeoffs and limited comparability across datasets. Here, we present a method based on statistical hypothesis testing on curves, which provides control of the false discovery rate. We apply it to several datasets probing epigenetic drugs and a metabolite. This leads us to detect off-target drug engagement, including the finding that the HDAC8 inhibitor PCI-34051 and its analog BRD-3811 bind to and inhibit leucine aminopeptidase 3. An implementation is available as an R package from Bioconductor ( https://bioconductor.org/packages/TPP2D ). We hope that our method will facilitate prioritizing targets from thermal profiling experiments.

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy.

Cancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery.

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery.

Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors.

The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool "housekeeping" enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.

Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1.

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit-to-lead development.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability.

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.

Experimental study on the influence of facial muscle activity on the facial mesostructure bones in rabbits.

UNLABELLED: Based on the functional matrix concept, scientists developed the hypothesis that soft tissue acting on certain bone pieces determines the process of facial growth. The possibility to modify muscle influence in the phase of facial development, or in postoperative of corrective surgery is of great preventive importance and it should be better investigated, since it could reduce the number and impact of these procedures. STUDY DESIGN: experimental in rabbits. AIM: to estimate the relevance of facial muscle activity on facial bones in lab rabbits. MATERIALS AND METHODS: 37 rabbits of two months of age were studied, divided in a study group and a control group, were followed up for a period of 4 months. The study group animals had their facial nerves cut at the cervical root in one side. The facial mesostructure of the animals was removed in block for later morphometric studies through computer graphics made out of the digital pictures of the specimens. Results were submitted for comparative statistical analysis. CONCLUSION: The lack of muscle activity in half of the face produces an ipsilateral shift of the facial mesostructure in developing rabbits.

Estudo experimental da influência da atividade muscular da face sobre o esqueleto da mesoestrutura facial em coelhos / Experimental study on the influence of facial muscle activity on the facial mesostructure bones in rabbits

A partir do conceito da matriz funcional, surgiu a hipótese de que são os tecidos moles atuando sobre determinada peça óssea que determinam o processo de crescimento facial. A possibilidade de modificar a influência muscular, seja na fase de desenvolvimento facial, seja em pós-operatórios de cirurgia corretiva é de grande importância preventiva e deveria ser mais bem investigada, uma vez que poderia subtrair o número e magnitude destes procedimentos. DESENHO DO ESTUDO: Experimental em coelhos. OBJETIVO: Estimar a relevância da atividade muscular sobre o esqueleto facial, em coelhos de experimentação, durante sua fase de desenvolvimento facial. MATERIAL E MÉTODO: Foram estudados 37 coelhos de 2 meses de idade, divididos em grupo de estudo e grupo controle e seguidos por um período de 4 meses. Os animais do grupo de estudo tiveram seus nervos faciais seccionados no seu ramo cervical unilateralmente. O esqueleto da mesoestrutura facial era retirado para estudo morfométrico por programa de computação gráfica em fotografias digitalizadas realizadas nas peças. Os resultados obtidos sofreram análise estatística comparativa. CONCLUSÃO: Ausência de atividade muscular em uma metade da face produz desvio lateral da mesoestrutura facial para o mesmo lado em coelhos em desenvolvimento.

Based on the functional matrix concept, scientists developed the hypothesis that soft tissue acting on certain bone pieces determines the process of facial growth. The possibility to modify muscle influence in the phase of facial development, or in postoperative of corrective surgery is of great preventive importance and it should be better investigated, since it could reduce the number and impact of these procedures. STUDY DESIGN: experimental in rabbits. AIM: to estimate the relevance of facial muscle activity on facial bones in lab rabbits. MATERIALS AND METHODS: 37 rabbits of two months of age were studied, divided in a study group and a control group, were followed up for a period of 4 months. The study group animals had their facial nerves cut at the cervical root in one side. The facial mesostructure of the animals was removed in block for later morphometric studies through computer graphics made out of the digital pictures of the specimens. Results were submitted for comparative statistical analysis. CONCLUSION: The lack of muscle activity in half of the face produces an ipsilateral shift of the facial mesostructure in developing rabbits.