General Installation of (4H)-Imidazolone cis-Amide Bioisosteres Along the Peptide Backbone.

Imidazolones represent an important class of heterocycles present in a wide range of pharmaceuticals, metabolites, and bioactive natural products and serve as the active chromophore in green fluorescent protein. Recently, imidazolones have received attention for their ability to act as a nonaromatic amide bond bioisotere which improves pharmacological properties. Herein, we present a tandem amidine installation and cyclization with an adjacent ester to yield (4H)-imidazolone products. Using amino acid building blocks, we can access the first examples of α-chiral imidazolones that have been previously inaccessible. Additionally, our method is amenable to on-resin installation which can be seamlessly integrated into existing solid-phase peptide synthesis protocols. Finally, we show that peptide imidazolones are potent cis-amide bond surrogates that preorganize linear peptides for head-to-tail macrocyclization. This work represents the first general approach to the backbone and side-chain insertion of imidazolone bioisosteres at various positions in linear and cyclic peptides.

Culturing the unculturables: strategies, challenges, and opportunities for gut microbiome study.

Metagenome sequencing techniques revolutionized the field of gut microbiome study. However, it is equipped with experimental and computational biases, which affect the downstream analysis results. Also, live microbial strains are needed for a better understanding of host-microbial crosstalks and for designing next-generation treatment therapies based on probiotic strains and postbiotic molecules. Conventional culturing methodologies are insufficient to get the dark gut matter on the plate; therefore, there is an urgent need to propose novel culturing methods that can fill the limitations of metagenomics. The current work aims to provide a consolidated evaluation of the available methods for host-microbe interaction with an emphasis on in vitro culturing of gut microbes using organoids, gut on a chip, and gut bioreactor. Further, the knowledge of microbial crosstalk in the gut helps us to identify core microbiota, and key metabolites that will aid in designing culturing media and co-culturing systems for gut microbiome study. After the deeper mining of the current culturing methods, we recommend that 3D-printed intestinal cells in a multistage continuous flow reactor equipped with an extended organoid system might be a good practical choice for gut microbiota-based studies.

Greater COVID-19 Severity and Mortality in Hospitalized Patients in the Delta-phase Compared to the Others: A Single Center Prospective Registry.

BACKGROUND AND OBJECTIVE: To compare clinical and laboratory features, and outcomes in the second COVID-19 phase (delta variant) with the first and third phases in India we performed a registry-based study. METHODS: Patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were recruited over the study period from March 2020 to July 2022. In the first phase (wild type, March-December 2020) of the 7,476 suspected, 1,395 (18.7%) were positive and 863 (61.8%) were hospitalized, in the second phase (delta, January-July 2021) out of 8,680 suspected, 1,641 (19.4%) tested positive and 388 (23.6%) were hospitalized, and in the third phase (omicron, January-July 2022) out of 5,188 suspected patients, 886 (17.1%) tested positive and 94 (10.6%) were hospitalized. We compared details of admission clinical and laboratory features and in-hospital management and outcomes in the three phases. RESULTS: A total of 2,352 patients were recruited. The majority of the patients were men, aged <45 years were 20% and about 20% of patients had hypertension, diabetes, and cardiovascular diseases. Patients in the second phase had significantly more cough, fever, shortness of breath, and lower oxygen saturation (SpO2) at admission and also had more lymphopenia, C-reactive proteins (CRPs), interleukin-6, ferritin, lactic dehydrogenase, and transaminases than patients in the other two phases. In the second vs the first and third phases, the requirement of supplementary oxygen (47.9 vs 33.1 and 23.4%), proning (89.2 vs 37.1 and 5.3%), high flow nasal oxygen (15.7 vs 8.71 and 5.3%), noninvasive ventilation (14.4 vs 9.1 and 11.7%), invasive ventilation (16.2 vs 9.1 and 9.6%), steroids (94.1 vs 83.4 and 37.2%), remdesivir (91.2 vs 73.8 and 39.4%), and anticoagulants (94.3 vs 83.0 and 61.7%) was significantly more (p < 0.001). The median length of stay in days [interquartile range (IQR)] was longer in the second phase [8 (6-10)] vs the first [7 (5-10)] and the third phase [4 (3-6) days]. The intensive care unit (ICU) stay in the second phase [9 (5-13) days] was also significantly more than the first [6 (2-10)] and third [0 (0-3)] phases (p <0.001). Overall, in-hospital deaths occurred in 176 patients (12.8%). Deaths were significantly higher in the second phase (19.3%), compared to the first (11.0%) and the third (3.3%) phases (p <0.01). We also observed that greater disease severity at presentation was associated with higher mortality in all the phases. CONCLUSION: This study shows that COVID-19 patients that were hospitalized in the second (delta) phase of the epidemic had more severe disease compared to the first and third phases. In the second phase of patients, there was a significantly higher duration of hospitalization, ICU hospitalization, greater oxygen requirement, noninvasive and invasive ventilatory support, and more deaths.

Design, synthesis, and applications of ring-functionalized histidines in peptide-based medicinal chemistry and drug discovery.

Modified and synthetic α-amino acids are known to show diverse applications. Histidine, which possesses numerous applications when subjected to synthetic modifications, is one such amino acid. The utility of modified histidines varies widely from remarkable biological activities to catalysis, and from nanotechnology to polymer chemistry. This renders histidine residue an important place in scientific research. Histidine is a well-studied scaffold and constitutes the active site of various enzymes catalyzing important reactions in the biological systems. A rational modification in histidine structure with a distinctly developed protocol extensively changes its physical and chemical properties. The utilization of modified histidines in search of potent, target selective and proteostable scaffolds is vital in the development of bioactive peptides with enhanced drug-likeliness. This review is a compilation and analysis of reported side-chain ring modifications at histidine followed by applications of ring-modified histidines in the synthesis of various categories of bioactive peptides and peptidomimetics.

Peptide-based drug discovery: Current status and recent advances.

The progressive development of peptides from reaction vessels to life-saving drugs via rigorous preclinical and clinical assessments is fascinating. Peptide therapeutics have gained momentum with the evolution of techniques in peptide chemistry, such as microwave irradiation in solid- and solution-phase synthesis, ligation chemistry, recombinant synthesis, and amalgamation with synthetic tools, including metal catalysis. Diverse emerging technologies, such as DNA-encoded libraries (DELs) and display techniques, are changing the status quo in the discovery of peptide therapeutics. In this review, we analyzed US Food and Drug Administration (FDA)-approved peptide drugs and those in clinical trials, highlighting recent advances in peptide-based drug discovery.

Synthetic amino acids-derived peptides target Cryptococcus neoformans by inducing cell membrane disruption.

We investigated synthetic amino acid-based approach to design short peptide-based antibiotics. Tautomerically restricted, amphiphilic 1-aryl-l-histidines along with hydrophobic tryptophan were utilized to synthesize the designed peptides. l-Trp-l-His(1-biphenyl)-NHBzl (12e, IC50 = 1.91 µg/mL; MIC = 3.46 µg/mL) and l-His[1-(4-n-butylphenyl)]-l-Trp-l-His[1-(4-n-butylphenyl)]-NHBzl (16d, IC50 = 1.36 µg/mL; MIC = 2.46 µg/mL) produced potency against Cryptococcus neoformans. Peptides with moderate antibacterial activities (IC50s = 4.40-8.80 µg/mL) were also identified. The mechanism of action and cellular changes revealed that membrane disruption due to interactions of the positively charged peptides with the negatively charged membrane of the cryptococcal cells result in permeabilization, leading to pore formation. The internal localization of the peptides instigated the interactions with DNA causing fragmentation of the genetic material, which together with membrane disruption led to cell death. Flow cytometric analysis points to cells death by apoptotic pathway. Time kill kinetics and synergistic study confirmed the fungicidal nature and synergism with amphotericin B.

A General Strategy to Install Amidine Functional Groups Along the Peptide Backbone.

Amidines are a structural surrogate for peptide bonds, yet have received considerably little attention in peptides due to limitations in existing methods to access them. The synthetic strategy developed in this study represents the first robust and general procedure for the introduction of amidines into the peptide backbone. We exploit and further develop the utility and efficiency of thioimidate protecting groups as a means to side-step reactivity that ultimately renders existing methods unsuitable for the installation of amidines along the main-chain of peptides. This work is significant because it describes a generally applicable path to access unexplored peptide designs and architectures for new therapeutics made possible by the unique properties of amidines.

Association of health insurance status with coronary risk factors, coronary artery disease, interventions and outcomes in India.

Objective: Coronary artery disease (CAD) related hospitalization and interventions are associated with catastrophic out-of-pocket health expenditure in India. To evaluate differences in risk factors, disease severity, management and outcomes in uninsured vs insured CAD patients we performed a study. Methods: Successive CAD patients who underwent percutaneous intervention (PCI) at our centre were enrolled from January 2018 to June 2021. Clinical, angiographic and intervention data were periodically uploaded in the American College of Cardiology CathPCI platform. Descriptive statistics are reported. Results: 4672 CAD patients (men 3736, women 936) were included; uninsured were 2166 (46%), government insurance was in 1635 (36%) and private insurance in 871 (18%). Mean age was 60.1 ± 11 years, uninsured <50y were 21.6% vs 14.0% and 20.3% with government and private insurance. Among the uninsured prevalence of raised total and non-HDL cholesterol, any tobacco use, ST-elevation myocardial infarction (STEMI) and ejection fraction <30% were more (p < 0.01). In the STEMI group (n = 1985), rates of primary PCI were the highest in those with private insurance (38.7%) compared to others. Multivessel stenting (≥2 stents) was more among the insured patients. Median length of hospital stay was similar in the three groups. In-hospital mortality was slightly more in the uninsured (1.43%), compared to government (0.88) and privately insured (0.82) (p = 0.242). The cost of hospitalization and procedures was the highest among uninsured (US$ 2240, IQR 1877-2783) compared to government (US$ 1977, IQR 1653-2437) and privately insured (US$ 2013, IQR 1668-2633) (p < 0.001). Conclusions: Uninsured CAD patients in India are younger with more risk factors, acute coronary syndrome, STEMI, multivessel disease and coronary stenting compared to those with government or private insurance. The uninsured bear significantly greater direct costs with slightly greater mortality.

New structural classes of antimalarials.

Malaria remains a major vector borne disease claiming millions of lives worldwide due to infections caused by Plasmodium sp. Discovery and development of antimalarial drugs have previously been dominated majorly by single drug therapy. The malaria parasite has developed resistance against first line and second line antimalarial drugs used in the single drug therapy. This has drawn attention to find ways to alleviate the disease burden supplanted by combination therapy with multiple drugs to overcome drug resistance. Emergence of resistant strains even against the combination therapy has now mandated the revision of the current antimalarial pharmacotherapy. Research efforts of the past decade led to the discovery and identification of several new structural classes of antimalarial agents with improved biological attributes over the older ones. The following is a comprehensive review, addressed to the new structural classes of heterocyclic and natural compounds that have been identified during the last decade as antimalarial agents. Some of the classes included herein contain one or more pharmacophores amalgamated into a single bioactive scaffold as antimalarial agents, which act upon the conventional and novel targets.

Synthesis of a fluorinated pyronin that enables blue light to rapidly depolarize mitochondria.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Burden of Type 2 Diabetes and Associated Cardiometabolic Traits and Their Heritability Estimates in Endogamous Ethnic Groups of India: Findings From the INDIGENIUS Consortium.

To assess the burden of type 2 diabetes (T2D) and its genetic profile in endogamous populations of India given the paucity of data, we aimed to determine the prevalence of T2D and estimate its heritability using family-based cohorts from three distinct Endogamous Ethnic Groups (EEGs) representing Northern (Rajasthan [Agarwals: AG]) and Southern (Tamil Nadu [Chettiars: CH] and Andhra Pradesh [Reddys: RE]) states of India. For comparison, family-based data collected previously from another North Indian Punjabi Sikh (SI) EEG was used. In addition, we examined various T2D-related cardiometabolic traits and determined their heritabilities. These studies were conducted as part of the Indian Diabetes Genetic Studies in collaboration with US (INDIGENIUS) Consortium. The pedigree, demographic, phenotypic, covariate data and samples were collected from the CH, AG, and RE EEGs. The status of T2D was defined by ADA guidelines (fasting glucose ≥ 126 mg/dl or HbA1c ≥ 6.5% and/or use of diabetes medication/history). The prevalence of T2D in CH (N = 517, families = 21, mean age = 47y, mean BMI = 27), AG (N = 530, Families = 25, mean age = 43y, mean BMI = 27), and RE (N = 500, Families = 22, mean age = 46y, mean BMI = 27) was found to be 33%, 37%, and 36%, respectively, Also, the study participants from these EEGs were found to be at increased cardiometabolic risk (e.g., obesity and prediabetes). Similar characteristics for the SI EEG (N = 1,260, Families = 324, Age = 51y, BMI = 27, T2D = 75%) were obtained previously. We used the variance components approach to carry out genetic analyses after adjusting for covariate effects. The heritability (h2) estimates of T2D in the CH, RE, SI, and AG were found to be 30%, 46%, 54%, and 82% respectively, and statistically significant (P ≤ 0.05). Other T2D related traits (e.g., BMI, lipids, blood pressure) in AG, CH, and RE EEGs exhibited strong additive genetic influences (h2 range: 17% [triglycerides/AG and hs-CRP/RE] - 86% [glucose/non-T2D/AG]). Our findings highlight the high burden of T2D in Indian EEGs with significant and differential additive genetic influences on T2D and related traits.

Substrate Protein Interactions and Methylglyoxal Modifications Reduce the Aggregation Propensity of Human Alpha-A-Crystallin G98R Mutant.

The G98R mutation in αA-crystallin is associated with presenile cataract development in humans. Previous studies have indicated that mutant proteins altered structure, decreased stability, increased oligomeric size, loss of chaperone-like activity, and susceptibility to proteolysis could be contributing factors to cataract formation. To evaluate the effect of substrate protein interactions with the mutant protein on cataract formation, we have performed chaperone assays with alcohol dehydrogenase (ADH), citrate synthase (CS), and ßB2-crystallin (ßB2), and analyzed the reaction mixtures by multi-angle light scattering (MALS) analysis. It appears that αAG98R protein initially gets stabilized upon interaction with substrate proteins. Analysis of the chaperone-client protein complexes revealed that wild-type αA-crystallin interacts with substrate proteins to form compact complexes leading to a slight increase in oligomeric mass, whereas αAG98R forms less compact and high molecular weight complexes with the substrate, and the resulting complexes continue to increase in size over time. As a result, the soluble complexes formed initially by the mutant protein begin to scatter light and precipitate. We found that the stability and chaperone activity of the αAG98R can be improved by modifying the protein with low concentrations (50 µM) of methylglyoxal (MGO). Incubation of αAG98R protein (1 mg/ml) under aseptic conditions for 30 days at 37°C resulted in precipitation of the mutant protein. In contrast, mutant protein incubations carried out with 50 µM MGO remained soluble and transparent. SDS-PAGE analysis showed gradual autolysis of the mutant protein in the absence of MGO. The average molar mass of the mutant protein oligomers changed from 7,258 ± 12 kDa to 3,950 ± 08 kDa within 60 min of incubation with MGO. There was no further significant change in the molar mass of mutant protein when tested on day 7 of MGO treatment. Our data suggest that the initial stabilization of αAG98R by substrate proteins could delay congenital cataracts' appearance, and the uncontrolled long-term interaction amongst mutant subunits and substrate proteins could be the rationale behind presenile cataracts formation. The results also demonstrate the potential benefit of low concentrations of MGO in stabilizing mutant chaperone protein(s).

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54-61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54-61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21-28 residues from the activated αBΔ54-61 (to get αBΔ21-28, Δ54-61) on the structure-function of recombinant αBΔ21-28, Δ54-61. The αBΔ21-28, Δ54-61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21-28, ∆54-61 was found to prevent ß-amyloid (Aß1-42) fibril formation in vitro and suppressed Aß1-42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54-61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54-61 in oxidatively stressed cells. Our study shows that the residues 21-28 and 54-61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21-28 residues further potentiates the activated αBΔ54-61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21-28, Δ54-61.

Modulating ß-arrestin 2 recruitment at the δ- and µ-opioid receptors using peptidomimetic ligands.

µ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit ß-arrestins, which correlate with certain adverse effects at µ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced ß-arrestin recruitment efficacy through both the δ-opioid and µ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand-receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased µ/δ opioid agonist pharmacological profiles.

Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Previously, we showed that the removal of the 54-61 residues from αB-crystallin (αBΔ54-61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone-substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54-61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54-61 protein.

Modified histidine containing amphipathic ultrashort antifungal peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent anticryptococcal activity.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37-9.66 µg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC50 = 0.37 µg/mL, MIC = 0.63 µg/mL) related to amphotericin B, without any cytotoxic effects up to 10 µg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead.

Structural and mechanistic insights into the inhibition of amyloid-ß aggregation by Aß39-42 fragment derived synthetic peptides.

The inhibition of amyloid-ß (Aß) aggregation is a promising approach towards therapeutic intervention for Alzheimer's disease (AD). Thirty eight tetrapeptides based upon Aß39-42C-terminus fragment of the parent Aß peptide were synthesized. The sequential replacement/modification employing unnatural amino acids imparted scaffold diversity, augmented activity, enhanced blood brain barrier permeability and offered proteolytic stability to the synthetic peptides. Several peptides exhibited promising protection against Aß aggregation-mediated-neurotoxicity in PC-12 cells at doses ranged between 10 µM and 0.1 µM, further confirmed by the thioflavin-T fluorescence assay. CD study illustrate that these peptides restrict the ß-sheet formation, and the non-appearance of Aß42 fibrillar structures in the electron microscopy confirm the inhibition of Aß42 aggregation. HRMS and ANS fluorescence spectroscopic analysis provided additional mechanistic insights. Two selected lead peptides 5 and 16 depicted enhanced blood-brain penetration and stability against serum and proteolytic enzyme. Structural insights into ligand-Aß interactions on the monomeric and proto-fibrillar units of Aß were computationally studied. Promising inhibitory potential and short sequence of the lead peptides offers new avenues for the advancement of peptide-derived therapeutics for AD.

Effect of C-terminus amidation of Aß39-42 fragment derived peptides as potential inhibitors of Aß aggregation.

The C-terminus fragment (Val-Val-Ile-Ala) of amyloid-ß is reported to inhibit the aggregation of the parent peptide. In an attempt to investigate the effect of sequential amino-acid scan and C-terminus amidation on the biological profile of the lead sequence, a series of tetrapeptides were synthesized using MW-SPPS. Peptide D-Phe-Val-Ile-Ala-NH2 (12c) exhibited high protection against ß-amyloid-mediated-neurotoxicity by inhibiting Aß aggregation in the MTT cell viability and ThT-fluorescence assay. Circular dichroism studies illustrate the inability of Aß42 to form ß-sheet in the presence of 12c, further confirmed by the absence of Aß42 fibrils in electron microscopy experiments. The peptide exhibits enhanced BBB permeation, no cytotoxicity along with prolonged proteolytic stability. In silico studies show that the peptide interacts with the key amino acids in Aß, which potentiate its fibrillation, thereby arresting aggregation propensity. This structural class of designed scaffolds provides impetus towards the rational development of peptide-based-therapeutics for Alzheimer's disease (AD).

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors.

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit ß-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

Resistant Hypertension in Clinical Practice in India: Jaipur Heart Watch.

BACKGROUND: Hypertension is highly prevalent in India but frequency of resistant hypertension has not been well studied. METHODS: We performed a registry-based study at a single center in patients with primary diagnosis of hypertension (n=3073). Details of co-morbidities, medications and blood pressure (BP) control were obtained. Patients with coronary heart disease, cerebrovascular disease and chronic kidney disease were excluded. Resistant hypertension was defined as uncontrolled hypertension (BP ≥140/90) with use of 3 drugs of which one was a diuretic, or any 4 drugs. RESULTS: Mean age of patients was 59±13 years, 47% were women and 26% <50y age. Diabetes was in 31.1%, hypothyroidism in 7.9% and chronic obstructive lung disease in 4.3%. The drugs prescribed were angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) in 61.5%, beta blockers in 49.8%, dihydropyridine calcium channel blockers (CCB) in 46.8%, ARB in 44.4%, diuretics in 32.1% ACEi in 13.4%, other CCBs in 2.6% and mineralocorticoid receptor antagonists (MRA) in 1.1%. One antihypertensive drug was prescribed in 27.4%, two in 41.2%, three in 18.6% and four or more in 5.4%. Prevalence of resistant hypertension using standard definition was 19.4% (95% confidence interval, CI, 18.0-20.8%). It was more in women (23.5%) vs men (15.7%) (p<0.001). Using the alternate definition the prevalence was 6.3% (95% CI 5.3-7.0%) and also more in women (6.9%) vs men (5.4%). Resistant hypertension was more common in patients >60 years (odds ratio 1.36, 95% CI 1.18-1.58) and women (odds ratio 1.64, 95% CI 1.37-1.97). CONCLUSION: Prevalence of resistant hypertension is high in a secondary-care practice in India. It is significantly greater among older patients and women.