Development of novel PEX5-PEX14 protein-protein interaction (PPI) inhibitors based on an oxopiperazine template.

Protein-protein interactions (PPIs) constitute an important but challenging class of molecular targets for small molecules. The PEX5-PEX14 PPI has been shown to play a critical role in glycosome biogenesis and its disruption impairs the metabolism in Trpanosoma parasites, eventually leading to their death. Therefore, this PPI is a potential molecular target for new drugs against diseases caused by Trypanosoma infections. Here, we report a new class of peptidomimetic scaffolds to target the PEX5-PEX14 PPI. The molecular design was based on an oxopiperazine template for the α-helical mimetics. A structural simplification along with modifications of the central oxopiperazine scaffold and addressing the lipophilic interactions led to the development of peptidomimetics that inhibit PEX5-TbPEX14 PPI and display cellular activity against T. b. brucei. This approach provides an alternative approach towards the development of trypanocidal agents and may be generally useful for the design of helical mimetics as PPI inhibitors.

Structure-based design, synthesis and evaluation of a novel family of PEX5-PEX14 interaction inhibitors against Trypanosoma.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

Validation of Mct8/Oatp1c1 dKO mice as a model organism for the Allan-Herndon-Dudley Syndrome.

OBJECTIVE: The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. METHODS: We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. RESULTS: We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. CONCLUSIONS: We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

Post-oral fat-induced satiation is mediated by endogenous CCK and GLP-1 in a fat self-administration mouse model.

Triacylglycerol is the most abundant dietary lipid, and a strong stimulator of satiation. Absorption of triacylglycerol in the small intestine occurs in the form of free fatty acids and 2-monoacylglycerol, a process known to trigger not only the release of cholecystokinin (CCK) but also glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). It remains controversial, however, whether endogenously released GLP-1 and PYY are required for fat-induced satiation. Using a self-administration model where mice are trained to self-administer Intralipid 30% intragastrically, we show that blocking the CCK1 receptors with intraperitoneal devazepide diminishes the post-oral satiation effect of ingested fat. Similarly, s.c. administration of a GLP-1 receptor antagonist with a prolonged half-life (Jant4-C16) also reduced the post-oral satiation effect of ingested fat. Importantly, coadministration of the GLP-1 antagonist together with devazepide increased fat self-infusions to a level equal to the combined blockade of each individual peptide action alone, indicating an additive effect of endogenous CCK and GLP-1 in fat satiation signaling. Blocking the PYY Y2 receptor did not further enhance the fat intake in devazepide-treated mice. Consistent with the above, we show that voluntary post-oral ingestion of fat increases CCK and GLP-1 plasma levels and is correlated positively with CCK and GLP-1 plasma concentrations. Taken together, our results support the role of endogenous GLP-1 in the regulation of fat intake and suggest that both CCK and GLP-1 are required for the fat satiation signaling.

The preliminary association study of osteopontin 707 C/T polymorphism with systemic lupus erythematosus in a Polish population.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by genetic, environmental, and still unknown factors which lead to deregulation of the immune system. Osteopontin (OPN) is a multifunctional glycoprotein, expressed in various cell types, and found to play key roles in immunity. OPN and variants of the OPN gene are involved in inflammatory conditions, however, their role in SLE are controversial. AIM: To investigate the frequency of single nucleotide polymorphism (SNP) rs1126616 (707 C/T) variants in the OPN gene and its associations with SLE manifestations in Polish patients. MATERIAL AND METHODS: The study population consisted of 83 SLE patients and 100 gender-, age- and ethnically matched healthy controls. DNA was extracted from whole blood samples using the standard procedure. Genotyping was performed by real-time polymerase chain reaction (RT-PCR). The association between clinical features of SLE and 707 C/T genotypes was determined. RESULTS: The mutant (CT, TT) genotypes were observed more frequently than the wild-type (CC) genotype in SLE patients compared to controls (p = 0.037). However, no association between 707 C/T variants and SLE clinical manifestations or laboratory parameters was found. CONCLUSIONS: The present data suggest that CT and TT genotypes of OPN 707 C/T SNP are associated with a higher SLE risk, but do not affect the clinical course of the disease in the Polish population.

Structural Refinement of Glucagon for Therapeutic Use.

Glucagon counters insulin's effects on glucose metabolism and serves as a rescue medicine in the treatment of hypoglycemia. Acute hypoglycemia, a common occurrence in insulin-dependent diabetes, is the central obstacle to correcting high blood glucose, a primary cause of long-term microvascular complications. As a result, there has been a resurgence of interest in improved glucagon therapy, including nonconventional liquid formulations, alternative routes of administration, and novel analogs with optimized biophysical properties. These options collectively minimize the complexity of glucagon delivery and enable its application in ways not feasible with conventional emergency rescue kits. These advances have indirectly promoted the integrated use of glucagon agonism with other hormones in a manner that runs counter to the long-standing pursuit of glucagon antagonism. This review summarizes novel approaches to glucagon optimization, methods with potential application to the broader family of therapeutic peptides, where biophysical challenges may be encountered.

The Correlation of Blood Parameters with Size in Cases of Neoplastic Tumor

Purpose: This study aimed to evaluate the impact of tumor volume on platelet counts (PLT) and mean platelet volume (MPV) and involve these parameters on overall survival. Methods: It is a retrospective study of 99 patients with lung cancer (confirmed histologically or cytologically). Sixty-six patients underwent radical operating treatment and 33 patients had only biopsies ­ due to the inoperable status of tumor According to the histopathology profile: non-small cell carcinoma ­ 23%, adenocarcinoma - 23 %, squamous - 36%, small cell carcinoma -11%, carcinoid ­ 6%. The overall survival was measured from the time of surgery to last observation or death. The tumor's size was established based on information from histopathology protocol by using model for the ellipsoid (V=4/3 π r abc). Results: KM median survival time after surgery was 20 months (95% C.I. = 16­42). The survival time depends significantly on: Tumor feature, MPV (p=0.03, p=0.04). Patients with normal PLT levels have longer survival time (median: 11 months) than thrombocytosis group (9.5) (p=0.6). Following both the PLT and MPV, a change-point that is equal to approximately 18.5 cm3 (approx. 3.3 cm in diameter) stands for a segmented relationship between tumor volume and analyzed blood indicators. Conclusions: After an overstepping of the change-point of tumor volume inflammatory processes start and they are associated with poor prognosis. MPV may be a valuable biomarker for the diagnosis and follow up of various types of carcinoma.

Nationwide survey on volunteers' training in hospice and palliative care in Poland.

BACKGROUND: Volunteers working in hospice and palliative care facilities in Poland undertake various activities which are performed in accordance with legal regulations and the individual policies of each hospice. The aim of this study was to explore the roles and training of volunteers working in hospice and palliative care settings. METHODS: A cross-sectional survey was carried out that investigated the services performed by volunteers and their preparation for work within residential hospices. Questionnaires were distributed to volunteers and hospice representatives, and the responses obtained underwent statistical analysis. PARTICIPANTS: A total of 180 volunteers and 28 hospice representatives from 29 residential hospices participated in this survey. RESULTS: All hospices surveyed were supported by volunteers. 79% of volunteers worked alongside patients and performed the following services: accompanying patients (76%), feeding patients (61%), cleaning rooms (48%), dressing and bathing (42%) and organising leisure time (40%). Fewer volunteers were involved in activities outside of patient support-for example, charity work and fundraising (34%), cleaning hospice buildings (23%) as well as providing information and education (22%). According to volunteers, prior to undertaking their duties, 64% participated in theoretical training and 37% took part in a practical course. The majority attended courses relating to general knowledge of hospice and palliative care (64%) and volunteer rights and duties (55%). CONCLUSIONS: Overall, proper training was an essential requirement needed to be fulfilled by volunteers, particularly when involved in direct patient support. Most volunteers were simultaneously involved in various areas of service; therefore, their training should be comprehensive.

Optimized GIP analogs promote body weight lowering in mice through GIPR agonism not antagonism.

OBJECTIVE: Structurally-improved GIP analogs were developed to determine precisely whether GIP receptor (GIPR) agonism or antagonism lowers body weight in obese mice. METHODS: A series of peptide-based GIP analogs, including structurally diverse agonists and a long-acting antagonist, were generated and characterized in vitro using functional assays in cell systems overexpressing human and mouse derived receptors. These analogs were characterized in vivo in DIO mice following acute dosing for effects on glycemic control, and following chronic dosing for effects on body weight and food intake. Pair-feeding studies and indirect calorimetry were used to survey the mechanism for body weight lowering. Congenital Gipr-/- and Glp1r-/- DIO mice were used to investigate the selectivity of the agonists and to ascribe the pharmacology to effects mediated by the GIPR. RESULTS: Non-acylated, Aib2 substituted analogs derived from human GIP sequence showed full in vitro potency at human GIPR and subtly reduced in vitro potency at mouse GIPR without cross-reactivity at GLP-1R. These GIPR agonists lowered acute blood glucose in wild-type and Glp1r-/- mice, and this effect was absent in Gipr-/- mice, which confirmed selectivity towards GIPR. Chronic treatment of DIO mice resulted in modest yet consistent, dose-dependent decreased body weight across many studies with diverse analogs. The mechanism for body weight lowering is due to reductions in food intake, not energy expenditure, as suggested by pair-feeding studies and indirect calorimetry assessment. The weight lowering effect was preserved in DIO Glp-1r-/- mice and absent in DIO Gipr-/- mice. The body weight lowering efficacy of GIPR agonists was enhanced with analogs that exhibit higher mouse GIPR potency, with increased frequency of administration, and with fatty-acylated peptides of extended duration of action. Additionally, a fatty-acylated, N-terminally truncated GIP analog was shown to have high in vitro antagonism potency for human and mouse GIPR without cross-reactive activity at mouse GLP-1R or mouse glucagon receptor (GcgR). This acylated antagonist sufficiently inhibited the acute effects of GIP to improve glucose tolerance in DIO mice. Chronic treatment of DIO mice with high doses of this acylated GIPR antagonist did not result in body weight change. Further, co-treatment of this acylated GIPR antagonist with liraglutide, an acylated GLP-1R agonist, to DIO mice did not result in increased body weight lowering relative to liraglutide-treated mice. Enhanced body weight lowering in DIO mice was evident however following co-treatment of long-acting selective individual agonists for GLP-1R and GIPR, consistent with previous data. CONCLUSIONS: We conclude that peptide-based GIPR agonists, not peptide-based GIPR antagonists, that are suitably optimized for receptor selectivity, cross-species activity, and duration of action consistently lower body weight in DIO mice, although with moderate efficacy relative to GLP-1R agonists. These preclinical rodent pharmacology results, in accordance with recent clinical results, provide definitive proof that systemic GIPR agonism, not antagonism, is beneficial for body weight loss.

Appraisal of Basic-Hemostatic Markers in Lung Cancer Patients During Follow-Up Care After Radiotherapy Treatment.

BACKGROUND The aim of this paper was to investigate the association between clinicopathological factors and the coagulation test in lung cancer patients during follow-up care after treatment. MATERIAL AND METHODS Ninety-five medical patients with histologically proven advanced lung carcinoma (LC) who had undergone radiotherapy were prospectively reviewed between January 2014 and December 2016. The study investigated the relationship between the biochemical results, the disease stage, and the survival rate in lung cancer patients. Post-treatment coagulation-based D-dimer (DD), fibrinogen (Fib), and complete blood count (CBC) were evaluated during the follow-up over a period of 2 years after treatment or until the patient's death. RESULTS An increase of D-dimer generates an increased chance of early death by approximately 0.03% per 1 D-dimer unit. In cases when the difference in the D-dimer concentration equals 1000, the risk of an early death increases by (1.00031000-1)×100%=35%. CONCLUSIONS High levels of D-dimer are associated with an advanced form of disease with metastasis and higher risk of early death in lung cancer patients.

Molecular elements in FGF19 and FGF21 defining KLB/FGFR activity and specificity.

OBJECTIVE: To signal, FGF19 and FGF21 require co-receptor ßKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. METHODS: We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. RESULTS: C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. CONCLUSIONS: We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.

Native Design of Soluble, Aggregation-Resistant Bioactive Peptides: Chemical Evolution of Human Glucagon.

Peptide-based therapeutics commonly suffer from biophysical properties that compromise pharmacology and medicinal use. Structural optimization of the primary sequence is the usual route to address such challenges while trying to maintain as much native character and avoiding introduction of any foreign element that might evoke an immunological response. Glucagon serves a seminal physiological role in buffering against hypoglycemia, but its low aqueous solubility, chemical instability, and propensity to self-aggregate severely complicate its medicinal use. Selective amide bond replacement with metastable ester bonds is a preferred approach to the preparation of peptides with biophysical properties that otherwise inhibit synthesis. We have recruited such chemistry in the design and development of unique glucagon prodrugs that have physical properties suitable for medicinal use and yet rapidly convert to native hormone upon exposure to slightly alkaline pH. These prodrugs demonstrate in vitro and in vivo pharmacology when formulated in physiological buffers that are nearly identical to native hormone when solubilized in conventional dilute hydrochloric acid. This approach provides the best of both worlds, where the pro-drug delivers chemical properties supportive of aqueous formulation and the native biological properties.

Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization.

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.

Vitamin D Receptor Gene BsmI Polymorphism in Polish Patients with Systemic Lupus Erythematosus.

The hormonally active form of vitamin D3, 1,25(OH)2D3 (calcitriol), exerts actions through VDR receptor, which acts as a transcriptional factor. Calcitriol is an immunomodulator that affects various immune cells, and several studies link it to many autoimmune diseases. BsmI polymorphism affects the level of VDR gene transcription, transcript stability, and posttranscriptional modifications. It seems to be related to the systemic lupus erythematosus (SLE). Our study examined the characteristics of VDR gene BsmI polymorphism in Polish SLE patients and their relationship with clinical manifestations of the disease. We genotyped 62 patients with SLE and 100 healthy controls using the real-time PCR. There were no differences observed in the frequency of BsmI genotypes in SLE patients and in the control group. There was no significant correlation between BsmI genotypes and clinical symptoms of SLE, but the AA genotype correlates with higher levels of antinuclear antibodies (ANA) in this group (r = 0.438; P = 0.002). A larger study examining BsmI and other VDR gene polymorphisms is needed. It may allow explaining differences in the clinical picture of the disease and choosing a personalized therapy.