Peptide Analogues Research Articles

Synthesis and Biological Studies of New Temporin A Analogs Containing Unnatural Amino Acids in Position 7.

(1) Background: Antimicrobial resistance is growing at an extreme pace and has proven to be an urgent topic, for research into alternative treatments. Such a prospective possibility is hidden in antimicrobial peptides because of their low to no toxicity, effectiveness at low concentrations, and most importantly their ability to be used for multiple treatments. This work was focused on the study of the effect of the modification in position 7 of Temporin A on its biological activity; (2) Methods: The targeted peptides were synthesized using Fmoc/Ot-Bu SPPS. The antibacterial activity of the analogs was determined using the broth microdilution method and disk-diffusion method. In vitro tests were performed to determine the cytotoxicity, phototoxicity, and antiproliferative activity of the peptide analogs on a panel of tumor and normal cell lines; (3) Results: All analogs except DTCit showed good antibacterial activity, with DTDab having the best activity according to the disk-diffusion method. However, DTCit had an acceptable cytotoxicity, combined with good selectivity against the test MCF-7 cell line; (4) Conclusions: The obtained results revealed the importance of the basicity and length of the side chain at position 7 in the Temporin A sequence for both tested activities.

Enterolyin S, a Polythiazole-containing Hemolytic Peptide from Enterococcus caccae.

The β-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides.

Evaluating the antimicrobial and anti-biofilm activity of three synthetic antimicrobial Citropin analogs and their ability to fight against Staphylococcus aureus and Staphylococcus pseudintermedius.

We developed three new analogs of the antimicrobial peptide (AMP) Citropin 1.1: DAN-1-13, AJP-1-1, and HHX-2-28, and tested their potential antimicrobial and antibiofilm activities against Staphylococcus aureus and S. pseudintermedius. Potential cytotoxic or hemolytic effects were determined using cultured human keratinocytes and erythrocytes to determine their safety. To assess the antimicrobial activity of each compound, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined against methicillin-resistant and methicillin-susceptible strains of S. aureus and S. pseudintermedius. Activity against newly formed and mature biofilms was determined in two clinical isolates using spectrophotometry and scanning electron microscopy (SEM). All three compounds exhibited antimicrobial and bactericidal activity against all studied S. aureus and S. pseudintermedius strains, with MICs ranging from 4-32μg ml-1 and MBCs ranging from 8-128μg ml-1. Subinhibitory concentrations of all compounds also showed ant-biofilm activity in the two tested isolates. All compounds exhibited limited cytotoxic and hemolytic activity. Novel analogs of Citropin 1.1 exhibit antimicrobial and bactericidal activities against S. aureus and S. pseudintermedius isolates and inhibit the biofilm formation of these bacteria.

Quantitative analysis of therapeutic peptides by CZE using multiple sample injection in hydrodynamically closed separation system.

Therapeutic peptides have emerged as an innovative and promising class of therapeutic compounds in modern medicine. Synthetic peptide analogs triptorelin and lanreotide are known for their pronounced clinical versatility and potency. In this study, we present the development and validation of novel methods based on capillary zone electrophoresis performed in hydrodynamically closed system (HCS) and paired with ultraviolet detection and repeated injection sample introduction. To the best of our knowledge, we developed the first capillary electrophoresis-based method for the determination of lanreotide, and concurrently, the first HCS method for the determination of triptorelin. Maximal separation efficiency and signal intensity were achieved using background electrolytes composed of 50mM formic acid with the addition of 0.05% (v/v) methyl-hydroxyethyl cellulose. The proposed methods exhibit favorable performance characteristics, namely, calibration curve (r2 exceeding 0.99), low limits of detection (0.25µg/mL in a water matrix and 0.5µg/mL in synthetic urine), acceptable precision (relative standard deviation ranging from 2.2% to 9.6% for intraday repeatability and between 5.2% and 14.9% for interday reproducibility), and accuracy (relative errors falling within the 91.1%-107.8% range). The method for triptorelin determination was then used for its quantification in a commercially available drug dosage form (powder for injection) and in spiked synthetic urine samples. The developed methods were also evaluated according to the novel blue applicability grade index, revealing their superior applicability. The results collectively point out the potential of the proposed methods for both quality control and clinical investigations.

The Role of the Unbinding Cycle on the Coordination Abilities of the Bi-Cyclopeptides toward Cu(II) Ions.

Bicyclic peptides have attracted the interest of pharmaceutical companies because of their remarkable properties, putting them on a new path in medicine. Their conformational rigidity improves proteolytic stability and leads to rapid penetration into tissues via any possible route of administration. Moreover, elimination of renal metabolism is of great importance, for example, for people with a history of liver diseases. In addition, each ring can function independently, making bicyclic peptides extremely versatile molecules for further optimization. In this paper, we compared the potentiometric and spectroscopic properties studied by UV-vis, MCD, and EPR of four synthetic analogues of the bi-cyclic peptide c(PKKHP-c(CFWKTC)-PKKH) (BCL). In particular, we correlated the structural and spectral properties of complexes with coordinating abilities toward Cu(II) ions of MCL1 (Ac-PKKHPc(CFWKTC)PKKH-NH2) that contains the unbinding cycle and N- and C-terminal linear parts with two histidine residues, one per part; two monocyclic ligands containing one histidine residue, both in the N-terminal position, i.e., MCL2 (Ac-PKKHPc(CFWKTC)PKKS-NH2) and in the C-terminal position, i.e., MCL3 (Ac-PKKSPc(CFWKTC)PKKH-NH2), respectively; and the linear structure LNL (Ac-PKKHPSFWKTSPKKH-NH2). Potentiometric results have shown that the bicyclic structure promotes the involvement of the side chain imidazole donors in Cu(II) binding. On the other hand, the results obtained for the mono-cyclic analogues lead to the conclusion that the coordination of the histidine moiety as an anchoring group is promoted by its location in the peptide sequence further from the nonbinding cycle, strongly influencing the involvement of the amide donors in Cu(II) coordination.

False-Negative 68 Ga-FAPI PET/CT in Pediatric Thyroid Medullary Carcinoma Seen With 68 Ga-DOTATOC PET/CT and 18 F-FDG PET/CT.

In adults, 68 Ga-FAP inhibitor ( 68 Ga-FAPI) PET/CT outperforms 68 Ga-radiolabeled somatostatin analog peptides ( 68 Ga PET/CT) and 18 F-FDG PET/CT in detecting thyroid lesions. This is the case of a 13-year-old boy newly diagnosed with medullary thyroid cancer with high calcitonin level. 68 Ga PET/CT revealed the presence of only a primary thyroid lesion. Proven to be superior in detecting metastasis, 68 Ga-FAPI PET/CT was performed. The results came out negative for primary and potential metastatic lesions. This case sheds shed light on false-negatives reported in 68 Ga-FAPI PET/CT scans in pediatric patients, emphasizing the need for alternate radiotracers when a negative study is met.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

Computational Study of Designed Peptide Analogs against Pseudomonas aeruginosa as a Possible Inhibitor of LptD Membrane Protein

Background: Pseudomonas aeruginosa is a common cause of nosocomial infection worldwide and is responsible for 18-61% of deaths alone. The organism has gained resistance to many known antibiotics and evolved into a multidrug-resistant strain. The LptD outer membrane protein of P. aeruginosa is a special target of interest due to its role in outer membrane biogenesis. Objective: The study aimed to gain an insight into how mutations affect the overall properties of antimicrobial peptides and to identify novel peptide analogs against P. aeruginosa. Methods: The peptide analogs were designed and shortlisted based on physicochemical parameters and estimated free energy change in the current study. The docking studies for wild type and shortlisted peptides were performed against LptD protein of P. aeruginosa. The toxicity, allergenicity, and solubility analyses of peptide analogs with high binding affinity to LptD were also conducted. Results: The molecular docking results indicated that peptide analogs 523M26, 523M29, 523M34, and 523M35 for AP00523 (wild type); 608M12, 608M13, 608M19, 608M31, 608M32, 608M39, and 608M43 for AP00608 (wild type); and 2858M25 for AP02858 (wild type) bound effectively with LptD membrane than their wild types. The toxicity, allergenicity, and solubility analyses revealed all these peptide analogs to be nontoxic, non-allergen, and have good water solubility. Conclusion: The binding energies predicted 523M26, 608M39, and 2858M25 bind effectively to LptD proteins than their wild type. Based on docking analysis, it was further predicted that 608M39 has an estimated binding affinity greater than L27-11, which is a known peptidomimetic inhibitor of the LptD protein.

The Development of CDC25A-Derived Phosphoseryl Peptides That Bind 14-3-3ε with High Affinities.

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.

Peptides Are Cardioprotective Drugs of the Future: The Receptor and Signaling Mechanisms of the Cardioprotective Effect of Glucagon-like Peptide-1 Receptor Agonists.

The high mortality rate among patients with acute myocardial infarction (AMI) is one of the main problems of modern cardiology. It is quite obvious that there is an urgent need to create more effective drugs for the treatment of AMI than those currently used in the clinic. Such drugs could be enzyme-resistant peptide analogs of glucagon-like peptide-1 (GLP-1). GLP-1 receptor (GLP1R) agonists can prevent ischemia/reperfusion (I/R) cardiac injury. In addition, chronic administration of GLP1R agonists can alleviate the development of adverse cardiac remodeling in myocardial infarction, hypertension, and diabetes mellitus. GLP1R agonists can protect the heart against oxidative stress and reduce proinflammatory cytokine (IL-1β, TNF-α, IL-6, and MCP-1) expression in the myocardium. GLP1R stimulation inhibits apoptosis, necroptosis, pyroptosis, and ferroptosis of cardiomyocytes. The activation of the GLP1R augments autophagy and mitophagy in the myocardium. GLP1R agonists downregulate reactive species generation through the activation of Epac and the GLP1R/PI3K/Akt/survivin pathway. The GLP1R, kinases (PKCε, PKA, Akt, AMPK, PI3K, ERK1/2, mTOR, GSK-3β, PKG, MEK1/2, and MKK3), enzymes (HO-1 and eNOS), transcription factors (STAT3, CREB, Nrf2, and FoxO3), KATP channel opening, and MPT pore closing are involved in the cardioprotective effect of GLP1R agonists.

The Synthetic Surfactant CHF5633 Restores Lung Function and Lung Architecture in Severe Acute Respiratory Distress Syndrome in Adult Rabbits

PurposeAcute respiratory distress syndrome (ARDS) is a major cause of hypoxemic respiratory failure in adults. In ARDS extensive inflammation and leakage of fluid into the alveoli lead to dysregulation of pulmonary surfactant metabolism and function. Altered surfactant synthesis, secretion, and breakdown contribute to the clinical features of decreased lung compliance and alveolar collapse. Lung function in ARDS could potentially be restored with surfactant replacement therapy, and synthetic surfactants with modified peptide analogues may better withstand inactivation in ARDS alveoli than natural surfactants.MethodsThis study aimed to investigate the activity in vitro and the bolus effect (200 mg phospholipids/kg) of synthetic surfactant CHF5633 with analogues of SP‐B and SP‐C, or natural surfactant Poractant alfa (Curosurf®, both preparations Chiesi Farmaceutici S.p.A.) in a severe ARDS model (the ratio of partial pressure arterial oxygen and fraction of inspired oxygen, P/F ratio ≤ 13.3 kPa) induced by hydrochloric acid instillation followed by injurious ventilation in adult New Zealand rabbits. The animals were ventilated for 4 h after surfactant treatment and the respiratory parameters, histological appearance of lung parenchyma and levels of inflammation, oxidative stress, surfactant dysfunction, and endothelial damage were evaluated.ResultsBoth surfactant preparations yielded comparable improvements in lung function parameters, reductions in lung injury score, pro-inflammatory cytokines levels, and lung edema formation compared to untreated controls.ConclusionsThis study indicates that surfactant replacement therapy with CHF5633 improves lung function and lung architecture, and attenuates inflammation in severe ARDS in adult rabbits similarly to Poractant alfa. Clinical trials have so far not yielded conclusive results, but exogenous surfactant may be a valid supportive treatment for patients with ARDS given its anti-inflammatory and lung-protective effects.

Insights into Molecular Interactions between a GAPDH-Related Fish Antimicrobial Peptide, Analogs Thereof, and Bacterial Membranes.

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.

Amplifying endogenous stem cell migration for in situ bone tissue formation: Substance P analog and BMP mimetic peptide-loaded click-crosslinked hyaluronic acid hydrogel

Endogenous stem cell-driven in situ bone tissue formation has recently garnered increasing attention. Therefore, our study sought to refine methods to enhance the migration and subsequent osteogenic differentiation of these cells. Our innovative approach involves using an injectable hydrogel that combines click cross-linking sites and a BMP-2 mimetic peptide (BP) with hyaluronic acid (HA). This injectable formulation, hereinafter referred to as SPa + Cx-HA-BP, incorporates a substance P analog peptide (SPa) with Cx-HA-BP, proving versatile for in vitro and in vivo applications without cytotoxicity. The controlled release of SPa creates a gradient that guides endogenous stem cells towards the Cx-HA scaffold from specific tissue niches. Both Cx-HA and SPa+Cx-HA induced minimal changes in the expression of genes associated with osteogenic differentiation. In contrast, these genes were robustly induced by both SPa + Cx-HA+BP and SPa + Cx-HA-BP, in which BP was respectively integrated via physical and chemical methods. Remarkably, chemically incorporating BP (Cx-HA-BP) resulted in 4–9 times higher osteogenic gene expression than physically mixed BP in Cx-HA+BP. This study validates the role of SPa role in guiding endogenous stem cells toward the hydrogel and underscores the substantial impact of sustained BP presence within the hydrogel. Collectively, our findings offer valuable insights for the development of innovative strategies to promote endogenous stem cell-based tissue regeneration. The developed hydrogel effectively guides stem cells from their natural locations and facilitates sustained osteogenic differentiation, thus holding great promise for applications in regenerative medicine.

Site-Specifically Modified Peptide Inhibitors of Protein Tyrosine Phosphatase 1B and T-Cell Protein Tyrosine Phosphatase with Enhanced Stability and Improved In Vivo Long-Acting Activity.

Protein tyrosine phosphatase 1B (PTP1B) and TC-PTP can function in a coordinated manner to regulate diverse biological processes including insulin and leptin signaling, T-cell activation, and tumor antigen presentation, which makes them potential targets for several therapeutic applications. We have previously demonstrated that the lipidated BimBH3 peptide analogues were a new class of promising PTP1B inhibitors with once-weekly antidiabetic potency. Herein, we chemically synthesized two series of BimBH3 analogues via site-specific modification and studied their structure-activity relationship. The screened analogues S2, S6, A2-14, A2-17, A2-20, and A2-21 exhibited an improved PTP1B/TC-PTP dual inhibitory activity and achieved good stability in the plasma of mice and dogs, which indicated long-acting potential. In mouse models of type 2 diabetes mellitus (T2DM), the selected analogues S6, S7, A2-20, and A2-21 with an excellent target activity and plasma stability generated once-weekly therapeutic potency for T2DM at lower dosage (0.5 μmol/kg). In addition, evidence was provided to confirm the cell permeability and targeted enrichment of the BimBH3 analogues. In summary, we report here that site-specific modification and long fatty acid conjugation afforded cell-permeable peptidomimetic analogues of BimBH3 with enhanced stability, in vivo activity, and long-acting pharmacokinetic profile. Our findings could guide the further optimization of BimBH3 analogues and provide a proof-of-concept for PTP1B/TC-PTP targeting as a new therapeutic approach for T2DM, which may facilitate the discovery and development of alternative once-weekly anti-T2DM drug candidates.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it’s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure–function relations could be pivotal for optimizing future peptide design strategies.

Vosoritide treatment for children with hypochondroplasia: a phase 2 trial

Hypochondroplasia is a rare autosomal dominant skeletal dysplasia due to activating variants in FGFR3. It presents with disproportionate short stature with a wide range of clinical severity. There are currently no approved medications to treat short stature in children with hypochondroplasia. Vosoritide is a C-type natriuretic peptide analog that was recently approved for improving growth in children with achondroplasia. We aimed to evaluate the safety and efficacy of vosoritide in children with hypochondroplasia. We conducted a single-arm, phase 2, open-label trial at a single centre in the USA and enrolled 26 children with hypochondroplasia. The trial consists of a 6-month observation period to establish a baseline annualized growth velocity followed by a 12-month intervention period during which vosoritide is administered daily via subcutaneous injection at a dose of 15μg/kg/day. The trial's co-primary endpoints included the incidence of adverse events and the change from baseline in age-sex standardized annualized growth velocity and height standardized deviation score (SDS) after 12 months of treatment. This trial is registered with ClinicalTrials.gov (NCT04219007). Twenty-four participants with a mean age of 5.86 years received vosoritide therapy. The first participant was enrolled on August 4, 2020, and the final participant completed the 18-month trial on September 8, 2023. Vosoritide was well tolerated with no treatment-related serious adverse events. Injection site reactions occurred in 83.3% of participants. No participants discontinued therapy due to an adverse event. Annualized growth velocity increased by 2.26 standard deviations (SD) and height SDS increased by 0.36 SD during the treatment period versus the observation period. Hypochondroplasia specific height SDS increased by 0.38 SD. There was a 1.81cm/year increase in absolute annualized growth velocity. Vosoritide was safe and effective in increasing growth velocity in children with hypochondroplasia. Efficacy was similar to what has been reported in children with achondroplasia. This study was supported by an investigator-initiated grant from BioMarin Pharmaceutical.

Thermodynamic consequences of stapling side-chains on a peptide ligand using a lactam-bridge: A theoretical study on anti-angiogenic peptides targeting VEGF.

Peptides are promising therapeutic agents for various biological targets due to their high efficacy and low toxicity, and the design of peptide ligands with high binding affinity to the target of interest is of utmost importance in peptide-based drug design. Introducing a conformational constraint to a flexible peptide ligand using a side-chain lactam-bridge is a convenient and efficient method to improve its binding affinity to the target. However, in general, such a small structural modification to a flexible ligand made with the intent of lowering the configurational entropic penalty for binding may have unintended consequences in different components of the binding enthalpy and entropy, including the configurational entropy component, which are still not clearly understood. Toward probing this, we examine different components of the binding enthalpy and entropy as well as the underlying structure and dynamics, for a side-chain lactam-bridged peptide inhibitor and its flexible analog forming complexes with vascular endothelial growth factor (VEGF), using all-atom molecular dynamics simulations. It is found that introducing a side-chain lactam-bridge constraint into the flexible peptide analog led to a gain in configurational entropy change but losses in solvation entropy, solute internal energy, and solvation energy changes upon binding, pinpointing the opportunities and challenges in drug design. The present study features an interplay between configurational and solvation entropy changes, as well as the one between binding enthalpy and entropy, in ligand-target binding upon imposing a conformational constraint into a flexible ligand.

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor

BackgroundSingle-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides.ResultsThe water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.ConclusionsWe established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.