The efficacy outcomes in non-small cell lung cancer patients treated with PD axis inhibitor agents - a population-based study of the Vojvodina region.

Background: By 2021, the FDA approved the use of the drugs pembrolizumab and atezolizumab in the first-line treatment of patients with high positivity of programmed death ligand-1 (PD-L1) in locally advanced and metastatic non-small-cell-lung cancer (NSCLC). This approval was the result of statistically significant evidence from international, multicentric clinical studies that all reported increasing progression-free survival (PFS) and overall survival (OS) in these patients. Methods: In our study, we reported the demographic and clinical characteristics of 79 patients diagnosed with NSCLC with expression of PD-L1 ≥50% from January 2019 to December 2022 at the Institute for Pulmonary Diseases of Vojvodina, who received pembrolizumab therapy as the first-line treatment. Patients were divided according to the histological type of lung cancer as adenocarcinoma (ADC) or squamous cell carcinoma (SCC) of the lung. In 52 of the 79 patients, PFS and in 32 of them overall survival (censored OS) was shown according to the histological type of tumor, the tumor proportion score (TPS) of PDL-1 expression, and the metastatic status within the Tumor Nodes Metastasis (TNM) disease classification. Independent factors of death outcome were shown by multivariable proportional hazard regression analysis. Results: The study included 79 patients diagnosed with NSCLC with an expression of PD-L1 ≥50%, 50 (63.3%) patients with ADC, and 29 (36.7%) patients with SCC, whose 55 (69.6%) PDL-1 expression was obtained from broncho biopsy (BB). The majority of patients, 49 (62%), had a TPS PD-L1 score of 51%-79%. Median, PFS for adenocarcinoma was 22 months and censored OS was 27 months, while for squamous cell carcinoma, median PFS was 12 months, and censored OS was 21 months. M1b disease stage, which was the most common in patients, had a PFS of 16 months and a censored OS of 18 months. Conclusion: Pembrolizumab monotherapy in patients with NSCLC in the fourth stage of the disease and with the positivity of the immune checkpoint protein TPS PD-L1 above 50% represents a safe therapy that allows a satisfactory period without disease progression and overall survival with acceptable treatment complications.

Assessment and Application of the Hear Score in Remote Emergency Medicine Outposts in Bosnia and Herzegovina.

Background and Objectives. In emergency departments, chest pain is a common concern, highlighting the critical importance of distinguishing between acute coronary syndrome and other potential causes. Our research aimed to introduce and implement the HEAR score, specifically, in remote emergency outposts in Bosnia and Herzegovina. Materials and Methods. This follow-up study conducted a retrospective analysis of a prospective cohort consisting of patients who were admitted to the remote emergency medicine outposts in Canton Sarajevo and Zenica from 1 November to 31 December 2023. Results. This study comprised 103 (12.9%) patients with low-risk HEAR scores and 338 (83.8%) with high-risk HEAR scores, primarily female (221, 56.9%), with a mean age of 63.5 ± 11.2). Patients with low-risk HEAR scores were significantly younger (50.5 ± 15.6 vs. 65.9 ± 12.1), had fewer smokers (p < 0.05), and exhibited a lower incidence of cardiovascular risk factors compared to those with high-risk HEAR scores. Low-risk HEAR score for prediction of AMI had a sensitivity of 97.1% (95% CI 89.9-99.6%); specificity of 27.3% (95% CI 22.8-32.1%); PPV of 19.82% (95% CI 18.67-21.03%), and NPV of 98.08% (95% CI 92.80-99.51%). Within 30 days of the admission to the emergency department outpost, out of all 441 patients, 100 (22.7%) were diagnosed with MACE, with AMI 69 (15.6%), 3 deaths (0.7%), 6 (1.4%) had a CABG, and 22 (4.9%) underwent PCI. A low-risk HEAR score had a sensitivity of 97.0% (95% CI 91.7-99.4%) and specificity of 27.3% (95% CI 22.8-32.1%); PPV of 25.5% (95% CI 25.59-28.37%); NPV of 97.14% (95% CI 91.68-99.06%) for 30-day MACE. Conclusions. In conclusion, the outcomes of this study align with existing research, underscoring the effectiveness of the HEAR score in risk stratification for patients with chest pain. In practical terms, the implementation of the HEAR score in clinical decision-making processes holds significant promise.

Impact of risk factors, early rehabilitation and management of lymphedema associated with breast cancer: a retrospective study of breast Cancer survivors over 5 years.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a potentially disabling and often irreversible consequence of breast cancer treatment, caused by the mechanical incompetence of the lymphatic system, resulting in reduced drainage capacity and functional overload due to an excessive volume of interstitial fluid surpassing the system's transport capacity in the arm. We wanted to determine the impact and explore the differences in independent risk factors for the occurrence of BCRL; incidence of BCRL over a five-year period at the Institute of Oncology Vojvodina in Sremska Kamenica and to answer the research question regarding the influence of the prehabilitation program on the overall incidence of BCRL during the observed five-year period. METHODS: From 2014 to 2018, a retrospective study was conducted at the Institute of Oncology of Vojvodina in Sremska Kamenica, analyzing female patients who had undergone breast cancer surgery. RESULTS: The study included 150 breast cancer patients who developed secondary lymphedema following surgery with the mean age of 59.2 ± 11.3 years. Fluctuations in hospitalization rates were observed over the five-year period, with the highest number of admissions in 2014 (24.0%) and a decline in 2018 (14.0%). The most common surgical procedure performed was left quadrantectomy (24.0%), followed by right quadrantectomy (20.0%) and left amputation (15.3%). The mean number of removed lymph nodes was 15.2 ± 6.1, with no statistically significant association between the number of removed lymph nodes and the manifestation of secondary lymphedema. The severity of secondary lymphedema varied based on patient age, with a higher incidence of moderate and severe lymphedema observed in patients aged 61 years and older. Patients who underwent radical surgery were more likely to experience severe lymphedema compared to those who had conservative surgery, although this difference was not statistically significant. CONCLUSION: In our study, the type of surgery, elapsed time since surgery, and the number of removed lymph nodes were not influencing factors for the occurrence of BCRL. However, concerning its severity, a greater number of systemic therapy modalities combined with radiotherapy were associated with a more frequent occurrence of mild and moderate BCRL. Also, the severity of BCRL varied among different age groups, with a higher incidence of moderate and severe lymphedema observed in patients aged 61 years and older. Ultimately, improving the quality of life for individuals affected by secondary lymphedema remains a crucial goal in the field of oncology.

Effective Differences between 2D and 3D Planned Brachytherapy in Lung Cancer: An Institutional Retrospective Study.

Background and Objectives: Advanced lung cancer is usually manifested by endoluminal tumor propagation, resulting in central airway obstruction. The objective of this study is to compare the high dose rate brachytherapy treatment outcomes in non-small-cell lung cancer (NSCLC) depending on the treatment planning pattern-two-dimension (2D) or three-dimension (3D) treatment planning. Materials and Methods: The study was retrospective and two groups of patients were compared in it (a group of 101 patients who underwent 2D planned high-dose-rate endobronchial brachytherapy (HDR-EBBT) in 2017/18 and a group of 83 patients who underwent 3D planned HDR-EBBT between January 2021 and June 2023). Results: In the group of 3D planned brachytherapy patients, there was a significant improvement in terms of loss of symptoms of bronchial obstruction (p = 0.038), but no improvement in terms of ECOG PS (European Cooperative Oncology Group Performance Status) of the patient (p = 0.847) and loss of lung atelectasis (if there was any at the beginning of the disease) (p = 0.781). Two-year overall survival and time-to-progression periods were similar for both groups of patients (p = 0.110 and 0.154). Fewer treatment complications were observed, and 91.4% were in 3D planned brachytherapy (BT) patients. Conclusions: Three-dimensionally planned HDR-EBBT is a suggestive, effective palliative method for the disobstruction of large airways caused by endobronchial lung tumor growth. Independent or more often combined with other types of specific oncological treatment, it certainly leads to the loss of symptoms caused by bronchial obstruction and the improvement of the quality of life of patients with advanced NSCLC. Complications of the procedure with 3D planning are less compared to 2D planned HDR-EBBT.

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

The Role of Resilience in the Relationship between Sociodemographic, Clinical Characteristics, and Social Support among Breast Cancer Patients in Serbia.

BACKGROUND: The management of breast cancer treatments within the limitations of family, social, and professional life is emotionally burdening and negatively affects physical, psychological, and social well-being, reducing the overall quality of life of patients and their families. METHODS: This cross-sectional descriptive-analytical study was conducted from March to August 2023 at the "Dr. Radivoj Simonovic" General Hospital in Sombor. A total of 236 breast cancer patients participated in this study. The research was conducted using the following instruments: a questionnaire on sociodemographic and clinical characteristics of patients, the Berlin Social-Support Scales-for assessing social support-and the Connor-Davidson Resilience Scale-for assessing resilience. This study aimed to determine the predictors and levels of social support and resilience of breast cancer patients. We also wanted to examine whether resilience is a mediator between patients' sociodemographic and clinical characteristics and levels of social support. RESULTS: The total average value of social support was 3.51 ± 0.63, while on the resilience scale, the respondents achieved a total average score of 52.2 ± 9.63. Perceived and actually received social support of breast cancer patients were positively correlated with resilience [p < 0.01], while no statistically significant correlations were found for the need for support and satisfaction. The sets of predictors can significantly predict their effects on all types of perceived social support (emotional social support: 9%; perceived instrumental social support: 9%) and all types of received social support (actually received emotional social support: 8%; actually received instrumental social support: 7%; actually received informational social support: 8%). There is a potential mediating role of resilience in relation to sociodemographic factors, clinical characteristics, and the need for support. CONCLUSION: This study confirms that a strong connection exists between social support and resilience. However, the analysis did not confirm the mediating role of resilience between the sociodemographic and clinical characteristics on the one hand and social support on the other.

HEART Score and Its Implementation in Emergency Medicine Departments in the West Balkan Region-A Pilot Study.

BACKGROUND: Chest pain represents a prevalent complaint in emergency departments (EDs), where the precise differentiation between acute coronary syndrome and alternative conditions assumes paramount significance. This pilot study aimed to assess the HEART score's implementation in West Balkan EDs. METHODS: A retrospective analysis was performed on a prospective cohort comprising patients presenting with chest pain admitted to EDs in Sarajevo, Zenica, and Belgrade between July and December 2022. RESULTS: A total of 303 patients were included, with 128 classified as low-risk based on the HEART score and 175 classified as moderate-to-high-risk. The low-risk patients exhibited younger age and a lower prevalence of cardiovascular risk factors. Laboratory and anamnestic findings revealed higher levels of C-reactive protein, ALT, and creatinine, higher rates of moderately to highly suspicious chest pain history, a greater number of cardiovascular risk factors, and elevated troponin levels in moderate-to-high-risk patients. Comparatively, among patients with a low HEART score, 2.3% experienced MACE, whereas those with a moderate-to high-risk HEART score had a MACE rate of 10.2%. A moderate-to-high-risk HEART score demonstrated a sensitivity of 91.2% (95%CI 90.2-93.4%) and specificity of 46.5% (95%CI 39.9-48.3%) for predicting MACE. CONCLUSION: This pilot study offers preliminary insights into the integration of the HEART score within the emergency departments of the West Balkan region.

Identification of SH2 Domain-Containing Protein 3C as a Novel, Putative Interactor of Dipeptidyl Peptidase 3.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent exopeptidase with broad specificity for four to eight amino acid residue substrates. It has a role in the regulation of oxidative stress response NRF2-KEAP1 pathway through the interaction with KEAP1. We have conducted stable isotope labeling by amino acids in a cell culture coupled to mass spectrometry (SILAC-MS) interactome analysis of TRex HEK293T cells using DPP3 as bait and identified SH2 Domain-Containing Protein 3C (SH2D3C) as prey. SH2D3C is one of three members of a family of proteins that contain both the SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain), named novel SH2-containing proteins (NSP). NSPs, including SH2D3C (NSP3), are adaptor proteins involved in the regulation of adhesion, migration, tissue organization, and immune response. We have shown that SH2D3C binds to DPP3 through its C-terminal Ras GEF-like domain, detected the colocalization of the proteins in living cells, and confirmed direct interaction in the cytosol and membrane ruffles. Computational analysis also confirmed the binding of the C-terminal domain of SH2D3C to DPP3, but the exact model could not be discerned. This is the first indication that DPP3 and SH2D3C are interacting partners, and further studies to elucidate the physiological significance of this interaction are on the way.

Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III.

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the Kd values for the binding of Zn2+, Cu2+, and Co2+ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the Kd values for the binding of these metals to an additional binding site. The structure of the catalytic metal's binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn2+, Cu2+, Co2+, and Mn2+ using the QM approach. The structures of the additional binding sites for the Zn2+ and Cu2+ were also identified, and MD simulations showed that two Cu2+ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn2+ ions bound to these sites.

Cervical Cancer Prevention Knowledge (Cckp-64) among Female Students in Novi Sad, Serbia during COVID-19 Pandemic.

Cervical cancer is a significant global health concern affecting young women, with over 500,000 new cases reported annually. This questionnaire-based study aimed to evaluate the knowledge of cervical cancer prevention among female students at the University of Novi Sad during the COVID-19 pandemic using the Cervical Cancer Knowledge Prevention-64 (CCKP-64) tool. The study sample consisted of 402 predominantly 20-22-year-old female students from either social or technical science faculties in urban environments. Results revealed that out of the 402 female students involved in the study, most had a good general knowledge of primary prevention of cervical cancer, with a correct answer rate ranging from 29.9 to 80.6%. On the contrary, only 63.4% of female students have heard about the vaccine against cervical cancer; 52.0% know that the vaccine exists in Serbia; and 31.8% know where to get vaccinated. Only a small proportion of students (9.7%) have encountered cervical cancer among their relatives/friends and think that the disease could affect them in the future (25.4%). Older students (>26 years) generally (p < 0.05) had better knowledge regarding distressing symptoms of cervical cancer, cytological examination and secondary prevention; however, it was also noted that a significant percentage of this age group reported not having received vaccinations (53.0%, p = 0.001). This study underscores the need for increased awareness and education about the HPV vaccine and secondary prevention among young women in Serbia. Future research should investigate knowledge and attitudes toward cervical cancer prevention in diverse populations to develop effective interventions and strategies. These findings have implications for public health policies in Serbia to promote cervical cancer prevention among young women.

COVID-19-Related Stress, Fear and Online Teaching Satisfaction among Nursing Students during the COVID-19 Pandemic.

The COVID-19 pandemic has had a significant impact on mental health, particularly among students, due to COVID-19-related fear and also the transition from traditional to online lectures. In this questionnaire-based study, the COVID-19 Stress Scales (CSS), the Fear of COVID-19 Scale (FCV-19S), and the Online Teaching Satisfaction Scale were used to assess COVID-19-related fear, stress, and overall satisfaction with online teaching during the COVID-19 pandemic among nursing students in Serbia. A total of 167 students participated in the study, whose mean age was 21.3 ± 5.3, and the majority of whom were female and first-year students. Overall, most students experienced moderate to extremely high COVID-19-related stress levels. Overall, first-year and fourth-year students scored significantly lower regarding the Xenophobia and Traumatic stress subscales than second-year and third-year students, whereas first-year students also scored significantly lower on the Danger and Contamination subscales. First-year students experienced less COVID-19-related fear compared to senior students. Students were reasonably satisfied with online teaching. A stratified program is needed to prevent further decline of students' mental health and to improve their adaptation through public, health, and educational changes.

Influence of Mutations of Conserved Arginines on Neuropeptide Binding in the DPP III Active Site.

Dipeptidyl peptidase III (DPP III), a zinc exopeptidase, is involved in the final steps of intercellular protein degradation and has a marked affinity for opioid peptides such as enkephalins and endomorphins. Recently, we characterized a number of neuropeptides as potential substrates and inhibitors of human DPP III and provided an explanation for their differential behavior. These studies prompted us to investigate the influence of the conserved R399 and R669 on neuropeptides binding to DPP III. Measuring kinetic parameters in inhibitory assays, we found that mutation of R669 to Ala or Met significantly reduced the inhibitory properties of the slow substrates tynorphin and valorphin, whereas the effects on binding of the good substrates Arg2-2NA and Leu-enkephalin were small. Molecular dynamics simulations of wild-type (WT) and mutant DPP III complexes with Leu-enkephalin, tynorphin, valorphin, and Arg2-2NA in conjunction with calculations of binding free energies revealed that the lower inhibitory potency of slow substrates in the R669A mutant can be explained by the lower binding affinity of tynorphin and the higher propensity of valorphin to hydrolyze in the mutant than in WT. The R399A mutation was shown to affect the binding and/or hydrolysis of both good and slow substrates, with the effects on Leu-enkephalin being the most pronounced.

Experimental and computational evaluation of dipeptidyl peptidase III inhibitors based on quinazolinone-Schiff's bases.

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that sequentially hydrolyzes biologically active peptides by cleaving dipeptides from their N-termini. Although its fundamental role is not been fully elucidated, human DPP III (hDPP III) has been recognized in several pathophysiological processes of interest for drug development. In this article 27 quinazolinone-Schiff's bases were studied for their inhibitory activity against hDPP III combining an in vitro experiment with a computational approach. The biochemical assay showed that most compounds exhibited inhibitory activity at the 100 µM concentration. The best QSAR model included descriptors from the following 2D descriptor groups: information content indices, 2D autocorrelations, and edge adjacency indices. Five compounds were found to be the most potent inhibitors with IC50 values below 10 µM, while molecular docking predicted that these compounds bind to the central enzyme cleft and interact with residues of the substrate binding subsites. Molecular dynamics simulations of the most potent inhibitor (IC50=0.96 µM) provided valuable information explaining the role of PHE109, ARG319, GLU327, GLU329, and ILE386 in the mechanism of the inhibitor binding and stabilization. This is the first study that gives insight into quinazolinone-Schiff's bases binding to this metalloenzyme.Communicated by Ramaswamy H. Sarma.

Neuropeptides, substrates and inhibitors of human dipeptidyl peptidase III, experimental and computational study - A new substrate identified.

Dipeptidyl peptidase III (DPP III) is a cytosolic, two-domain zinc-exopeptidase. It is widely distributed in mammalian tissues, where it's involved in the final steps of normal intracellular protein degradation. However, its pronounced affinity for some bioactive peptides (angiotensins, enkephalins, and endomorphins) suggests more specific functions such as blood pressure regulation and involvement in pain regulation. We have investigated several different neuropeptides as potential substrates and inhibitors of human DPP III. The binding affinities and kinetic data determined by isothermal titration calorimetry, in combination with measurements of enzyme inhibition identified the hemorphin-related valorphin, tynorphin, S-tynorphin, and I-tynorphin as the most potent inhibitors of DPP III (actually slow substrates), whereas hemorphin-4 proved to be the best substrate of all neuropeptides examined. In addition, we have shown that the neuropeptides valorphin, Leu-valorphin-Arg, and the opioid peptide ß-casomorphin, are DPP III substrates. The molecular modelling of selected peptides shows uniform binding to the lower domain ß-strand residues of DPP III via peptide backbone atoms, but also previously unrecognized stabilizing interactions with conserved residues of the metal-binding site and catalytic machinery in the upper domain. The computational data helped explain the differences between substrates that are hydrolyzed effectively and those hydrolysed slowly by DPP III.

Recognition of ATT Triplex and DNA:RNA Hybrid Structures by Benzothiazole Ligands.

Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines-group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles-group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles-group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study's main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.

Interdisciplinary Study of the Effects of Dipeptidyl-Peptidase III Cancer Mutations on the KEAP1-NRF2 Signaling Pathway.

Dipeptidyl peptidase III (DPP III) is associated with cancer progression via interaction with KEAP1, leading to upregulation of the KEAP1-NRF2 oxidative stress pathway. Numerous DPP III mutations have been found in human tumor genomes, and it is suggested that some of them may alter affinity for KEAP1. One such example is the DPP III-R623W variant, which in our previous study showed much higher affinity for the Kelch domain of KEAP1 than the wild-type protein. In this work, we have investigated the effects of this mutation in cultured cells and the effects of several other DPP III mutations on the stability of KEAP1-DPP III complex using an interdisciplinary approach combining biochemical, biophysical and molecular biology methods with computational studies. We determined the affinity of the DPP III variants for the Kelch domain experimentally and by molecular modeling, as well as the effects of the R623W on the expression of several NRF2-controlled genes. We confirmed that the R623W variant upregulates NQO1 expression at the transcriptional level. This supports the hypothesis from our previous study that the increased affinity of the R623W variant for KEAP1 leads to upregulation of the KEAP1-NRF2 pathway. These results provide a new perspective on the involvement of DPP III in cancer progression and prognosis.

Demystifying DPP III Catalyzed Peptide Hydrolysis-Computational Study of the Complete Catalytic Cycle of Human DPP III Catalyzed Tynorphin Hydrolysis.

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes peptides of varying sequence and size. Despite attempts to elucidate and narrow down the broad substrate-specificity of DPP III, there is no explanation as to why some of them, such as tynorphin (VVYPW), the truncated form of the endogenous heptapeptide spinorphin, are the slow-reacting substrates of DPP III compared to others, such as Leu-enkephalin. Using quantum molecular mechanics calculations followed by various molecular dynamics techniques, we describe for the first time the entire catalytic cycle of human DPP III, providing theoretical insight into the inhibitory mechanism of tynorphin. The chemical step of peptide bond hydrolysis and the substrate binding to the active site of the enzyme and release of the product were described for DPP III in complex with tynorphin and Leu-enkephalin and their products. We found that tynorphin is cleaved by the same reaction mechanism determined for Leu-enkephalin. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme.

Triarylborane Dyes as a Novel Non-Covalent and Non-Inhibitive Fluorimetric Markers for DPP III Enzyme.

Novel dyes were prepared by simple "click CuAAC" attachment of a triarylborane-alkyne to the azide side chain of an amino acid yielding triarylborane dye 1 which was conjugated with pyrene (dye 2) forming a triarylborane-pyrene FRET pair. In contrast to previous cationic triarylboranes, the novel neutral dyes interact only with proteins, while their affinity to DNA/RNA is completely abolished. Both the reference triarylborane amino acid and triarylborane-pyrene conjugate bind to BSA and the hDPP III enzyme with high affinities, exhibiting a strong (up to 100-fold) fluorescence increase, whereby the triarylborane-pyrene conjugate additionally retained FRET upon binding to the protein. Furthermore, the triarylborane dyes, upon binding to the hDPP III enzyme, did not impair its enzymatic activity under a wide range of experimental conditions, thus being the first non-covalent fluorimetric markers for hDPP III, also applicable during enzymatic reactions with hDPP III substrates.

Coumarin Derivatives Act as Novel Inhibitors of Human Dipeptidyl Peptidase III: Combined In Vitro and In Silico Study.

Dipeptidyl peptidase III (DPP III), a zinc-dependent exopeptidase, is a member of the metalloproteinase family M49 with distribution detected in almost all forms of life. Although the physiological role of human DPP III (hDPP III) is not yet fully elucidated, its involvement in pathophysiological processes such as mammalian pain modulation, blood pressure regulation, and cancer processes, underscores the need to find new hDPP III inhibitors. In this research, five series of structurally different coumarin derivatives were studied to provide a relationship between their inhibitory profile toward hDPP III combining an in vitro assay with an in silico molecular modeling study. The experimental results showed that 26 of the 40 tested compounds exhibited hDPP III inhibitory activity at a concentration of 10 µM. Compound 12 (3-benzoyl-7-hydroxy-2H-chromen-2-one) proved to be the most potent inhibitor with IC50 value of 1.10 µM. QSAR modeling indicates that the presence of larger substituents with double and triple bonds and aromatic hydroxyl groups on coumarin derivatives increases their inhibitory activity. Docking predicts that 12 binds to the region of inter-domain cleft of hDPP III while binding mode analysis obtained by MD simulations revealed the importance of 7-OH group on the coumarin core as well as enzyme residues Ile315, Ser317, Glu329, Phe381, Pro387, and Ile390 for the mechanism of the binding pattern and compound 12 stabilization. The present investigation, for the first time, provides an insight into the inhibitory effect of coumarin derivatives on this human metalloproteinase.

SHH-N non-canonically sustains androgen receptor activity in androgen-independent prostate cancer cells.

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.