Tuning Electrostatic and Hydrophobic Surfaces of Aromatic Rings to Enhance Membrane Association and Cell Uptake of Peptides.

Aromatic groups are key mediators of protein-membrane association at cell surfaces, contributing to hydrophobic effects and π-membrane interactions. Here we show electrostatic and hydrophobic influences of aromatic ring substituents on membrane affinity and cell uptake of helical, cyclic and cell penetrating peptides. Hydrophobicity is important, but subtle changes in electrostatic surface potential, dipoles and polarizability also enhance association with phospholipid membranes and cell uptake. A combination of fluorine and sulfur substituents on an aromatic ring induces microdipoles that enhance cell uptake of 12-residue peptide inhibitors of p53-HDM2 interaction and of cell-penetrating cyclic peptides. These aromatic motifs can be readily inserted into peptide sidechains to enhance their cell uptake.

Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc.

c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy. Here we reassess the challenges of directly targeting c-Myc, evaluate lessons learned from current inhibitors, and explore how future strategies such as miniaturisation of Omomyc and targeting E-box binding could facilitate translation of c-Myc inhibitors into the clinic.

Late-Stage Hydrocarbon Conjugation and Cyclisation in Synthetic Peptides and Proteins.

The conventional S-alkylation of cysteine relies upon using activated electrophiles. Here we demonstrate high-yielding and selective S-alkylation and S-lipidation of cysteines in unprotected synthetic peptides and proteins by using weak electrophiles and a Zn2+ promoter. Linear or branched iodoalkanes can S-alkylate cysteine in an unprotected 38-residue Myc peptide fragment and in a 91-residue miniprotein Omomyc, thus highlighting selective late-stage synthetic modifications. Metal-assisted cysteine alkylation is also effective for incorporating dehydroalanine into unprotected peptides and for peptide cyclisation via aliphatic thioether crosslinks, including customising macrocycles to stabilise helical peptides for enhanced uptake and delivery to proteins inside cells. Chemoselective and efficient late-stage Zn2+ -promoted cysteine alkylation in unprotected peptides and proteins promises many useful applications.

Fortified Coiled Coils: Enhancing Mechanical Stability with Lactam or Metal Staples.

Coiled coils (CCs) are powerful supramolecular building blocks for biomimetic materials, increasingly used for their mechanical properties. Here, we introduce helix-inducing macrocyclic constraints, so-called staples, to tune thermodynamic and mechanical stability of CCs. We show that thermodynamic stabilization of CCs against helix uncoiling primarily depends on the number of staples, whereas staple positioning controls CC mechanical stability. Inserting a covalent lactam staple at one key force application point significantly increases the barrier to force-induced CC dissociation and reduces structural deformity. A reversible His-Ni2+ -His metal staple also increases CC stability, but ruptures upon mechanical loading to allow helix uncoiling. Staple type, position and number are key design parameters in using helical macrocyclic templates for fine-tuning CC properties in emerging biomaterials.

A Novel Long-Range n to π* Interaction Secures the Smallest known α-Helix in Water.

The introduction of an amide bond linking side chains of the first and fifth amino acids forms a cyclic pentapeptide that optimally stabilizes the smallest known α-helix in water. The origin of the stabilization is unclear. The observed dependence of α-helicity on the solvent and cyclization linker led us to discover a novel long-range n to π* interaction between a main-chain amide oxygen and a uniquely positioned carbonyl group in the linker of cyclic pentapeptides. CD and NMR spectra, NMR and X-ray structures, modelling, and MD simulations reveal that this first example of a synthetically incorporated long-range n to π* CO⋅⋅⋅Cγ =Ο interaction uniquely enforces an almost perfect and remarkably stable peptide α-helix in water but not in DMSO. This unusual interaction with a covalent amide bond outside the helical backbone suggests new approaches to synthetically stabilize peptide structures in water.

Contiguous hydrophobic and charged surface patches in short helix-constrained peptides drive cell permeability.

Most protein-protein interactions occur inside cells. Peptides can inhibit protein-protein interactions but tend not to enter cells. We systematically compare cell permeability for 8-12 residue model peptides with helix-inducing lactam/hydrocarbon linkers between amino acid sidechains. Cell uptake increases when hydrophobic residues and lactam linkers (i, i + 4) form a contiguous hydrophobic surface patch. Uptake increases further when both hydrophobic and positively charged (but not neutral or negative) residues are clustered into like surface patches. Amphipathicity alone is however insufficient for cell uptake of acyclic sequences. Changing the linker from lactam to hydrocarbon further increases uptake, but also promotes cell lysis. Helicity, positive charge and amphipathicity together promote cell permeability. Most known bioactive helical peptides do not optimally cluster residues for amphipathicity and so are likely unoptimised for cell uptake.

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (Kd) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pKi of 8.6 ± 0.2 and Ki of 2.5 nM) and C3aR ligands TR16 (pKi of 6.8 ± 0.1 and Ki of 138 nM), BR103 (pKi of 6.7 ± 0.1 and Ki of 185 nM), BR111 (pKi of 6.3 ± 0.2 and Ki of 544 nM) and SB290157 (pKi of 6.3 ± 0.1 and Ki of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

Review stapling peptides using cysteine crosslinking.

Stapled peptides are an emerging class of cyclic peptide molecules with enhanced biophysical properties such as conformational and proteolytic stability, cellular uptake and elevated binding affinity and specificity for their biological targets. Among the limited number of chemistries available for their synthesis, the cysteine-based stapling strategy has received considerable development in the last few years driven by facile access from cysteine-functionalized peptide precursors. Here we present some recent advances in peptide and protein stapling where the side-chains of cysteine residues are covalently connected with a range of different crosslinkers affording bisthioether macrocyclic peptides of varying topology and biophysical properties. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 843-852, 2016.

Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions.

The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

Helix Nucleation by the Smallest Known α-Helix in Water.

Cyclic pentapeptides (e.g. Ac-(cyclo-1,5)-[KAXAD]-NH2 ; X=Ala, 1; Arg, 2) in water adopt one α-helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α-helicity at the C-terminal aspartate caused by torsional restraints imposed by the K(i)-D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water-soluble 2 was appended to N-, C-, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α-helicity, as calculated from CD spectra. From the C-terminus of peptides, 2 can nucleate at least six α-helical turns. From the N-terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0-9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5-25 residue peptides, which correspond to most helix lengths in protein-protein interactions.

Comparative α-helicity of cyclic pentapeptides in water.

Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 310-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide.

Two in ten patients seeking addiction treatment within São Paulo's Crackland report recent use of Synthetic Cannabinoids.

The profile investigation of crack cocaine dependents seeking addiction treatment within Sao Paulo's open drug use scene known as "Crackland" demonstrated that 20% of them reported consuming variations of Synthetic Cannabinoids. This equated to 1.054 patients in a span of four months (between 08/05/2023 to 08/09/2023). This preliminary finding is well-timed and holds significant value making it suitable for a briefer publication format, such as a letter, that can promptly disseminate this relevant information to the scientific community. Additional and complementary evidence expanding the discussion beyond the medical aspects of SC consumption is also provided. This figure is a clear indication of the shift in the population accessing this drug, now reaching the most vulnerable segment of our nation's population. The letter brings on further insights with a more social perspective, also discussing the intricate interplay between affordability, availability price and how criminal organizations are likely involved now with SC's control and distribution in Brazil.

Landscaping macrocyclic peptides: stapling hDM2-binding peptides for helicity, protein affinity, proteolytic stability and cell uptake.

Cyclic peptides that modulate protein-protein interactions can be valuable therapeutic candidates if they can be delivered intact to their target proteins in cells. Here we systematically compare the effects of different helix-inducing cyclization constraints on the capacity of a macrocyclic peptide component to confer α-helicity, protein-binding affinity, resistance to degradative proteases and cell uptake to a 12-residue peptide fragment of tumor suppressor protein p53. We varied the helix-inducing constraint (hydrocarbon, lactam, aliphatic or aromatic thioether, etc.) and the position of the cyclization linker (i to i + 4 or i to i + 7 bridges) in order to sculpt the macrocyclic size, stabilize its structure, and promote cell uptake. We find that rigidifying the macrocycle leads to higher alpha helicity, target affinity and proteolytic stability to different extents, whereas cell uptake of compounds shown here is mostly driven by hydrophobicity and aromaticity of the macrocycle.

Telemedicine Versus Face-to-Face Care in Ophthalmology: Costs and Utility Measures in a Real-World Setting.

OBJECTIVES: Advances in telemedicine offer a unique opportunity to expand access to the health system. Nevertheless, few studies have described the impact of telediagnosis implementation on health and economic outcomes. METHODS: An ophthalmology telediagnosis service (TeleOftalmo) was compared with traditional face-to-face care provided by the Brazilian public health system. For both groups, utility data were collected at 2 time points using the Visual Function Questionnaire-Utility Index instrument from interviews with 536 patients. The cost per patient encounter was analyzed according to the time-driven activity-based costing. Value analyses were conducted to ascertain whether and how telemedicine service has the potential to generate cost savings for the health system. RESULTS: Visual function-related quality of life did not differ significantly between TeleOftalmo and face-to-face care groups. Using the current model, the telemedicine service assisted an average of 1159 patients per month at a median cost per telediagnosis of Int$97 (interquartile range, Int$82-Int$119) versus Int$77 (interquartile range, Int$75-Int$80) for face-to-face care. If the telemedicine service was redesigned, considering the opportunities for improvement identified, it could operate at a cost of Int$53 per telediagnosis (a 31% cost savings) and could serve 3882 patients per month. CONCLUSIONS: This study demonstrates the potential value of a telemedicine service. There was no difference in patient-perceived utility between a telediagnostic ophthalmology service and face-to-face care by an eye specialist. TeleOftalmo has the potential to be a cost-saving strategy for the Brazilian health system and could be a template for implementation of telediagnostic services in other regions.

Dynamics and Determinants of SARS-CoV-2 RT-PCR Testing on Symptomatic Individuals Attending Healthcare Centers during 2020 in Bahia, Brazil.

RT-PCR testing data provides opportunities to explore regional and individual determinants of test positivity and surveillance infrastructure. Using Generalized Additive Models, we explored 222,515 tests of a random sample of individuals with COVID-19 compatible symptoms in the Brazilian state of Bahia during 2020. We found that age and male gender were the most significant determinants of test positivity. There was evidence of an unequal impact among socio-demographic strata, with higher positivity among those living in areas with low education levels during the first epidemic wave, followed by those living in areas with higher education levels in the second wave. Our estimated probability of testing positive after symptom onset corroborates previous reports that the probability decreases with time, more than halving by about two weeks and converging to zero by three weeks. Test positivity rates generally followed state-level reported cases, and while a single laboratory performed ~90% of tests covering ~99% of the state's area, test turn-around time generally remained below four days. This testing effort is a testimony to the Bahian surveillance capacity during public health emergencies, as previously witnessed during the recent Zika and Yellow Fever outbreaks.

Health-related quality of life associated with diabetic retinopathy in patients at a public primary care service in southern Brazil.

OBJECTIVE: This study aimed to establish the utility values of different health states associated with diabetic retinopathy in a Brazilian sample to provide input to model-based economic evaluations. METHODS: This cross-sectional study was performed in a sample of patients with type 2 diabetes mellitus (T2D) who underwent teleophthalmology screening at a primary care service from 2014 to 2016. Five diabetic retinopathy health states were defined: absent, non-sight-threatening, sight-threatening, and bilateral blindness. Utility values were estimated using the Brazilian EuroQol five dimensions (EQ-5D) tariffs. Descriptive statistics were calculated. Analysis of covariance was performed to adjust the utility values for potential confounders. RESULTS: The study included 206 patients. The mean (± standard deviation [SD]) utility value was 0.765 ± 0.19 (95% confidence interval [CI], 0.740-0.790). The adjusted mean utility value was 0.748 (95% CI, 0.698-0.798) in patients without diabetic retinopathy, 0.752 (95% CI, 0.679-0.825) in those with non-sight-threatening state, 0.628 (95% CI, 0.521-0.736) in those with sight-threatening state, and 0.355 (95% CI, 0.105-0.606) in those with bilateral blindness. A significant utility decrement was found between patients without diabetic retinopathy and those with a sight-threatening health state (0.748 vs. 0.628, respectively, p = 0.04). CONCLUSION: The findings suggest that a later diabetic retinopathy health state is associated with a decrement in utility value compared with the absence of retinopathy in patients with T2D. The results may be useful as preliminary input to model-based economic evaluations. Further research is needed to investigate the impact of diabetic retinopathy on health-related quality of life in a sample more representative of the Brazilian population.

Effects of extra virgin olive oil and pecans on plasma fatty acids in patients with stable coronary artery disease.

OBJECTIVE: The aim of this study was to determine the effects of a healthy diet supplemented with extra virgin olive oil or pecans on plasma fatty acids (PFAs) in patients with stable coronary artery disease (CAD). METHODS: Patients 40 to 80 y of age were randomized to one of three dietary interventions (allocation ratio 1: 1: 1): healthy diet based on guidelines (control group [CG]), healthy diet supplemented with 30 g/d of pecans (PNG), or a healthy diet supplemented with 30 mL/d of extra virgin olive oil (OOG). PFAs were identified at baseline and at the end of follow-up (12 wk), and correlations between dietary fatty acids intake, PFAs, and clinical biomarkers of the lipid profile were also assessed before and after the interventions. RESULTS: Among 149 participants included in the analysis (43 CG; 51 PNG; and 55 OOG), correlations were observed between food intake, PFAs, and lipid profile before and after interventions independent of statins used, but all were considered weak. At the end of the study, the OOG showed increased concentrations of oleic fatty acid independently of the type of statin in use (1.49%; 95% confidence interval, 0.08-2.89; P = 0.029); however, there were no significant differences between the groups regarding the final mean values of oleic fatty acid or in the other PFAs. CONCLUSIONS: In patients with stable CAD, there were no significant differences in PFAs after 12 wk according to dietary interventions evaluated.

Teaming-up nurses with ophthalmologists to expand the reach of eye care in a middle-income country: Validation of health data acquisition by nursing staff in a telemedicine strategy.

Telemedicine can be used to conduct ophthalmological assessment of patients, facilitating patient access to specialist care. Since the teleophthalmology models require data collection support from other health professionals, the purpose of our study was to assess agreement between the nursing technician and the ophthalmologist in acquisition of health parameters that can be used for remote analysis as part of a telemedicine strategy. A cross-sectional study was conducted with 140 patients referred to an ophthalmological telediagnosis center by primary healthcare doctors. The health parameters evaluated were visual acuity (VA), objective ophthalmic measures acquired by autorefraction, keratometry, and intraocular pressure (IOP). Bland-Altman plots were used to analyze agreement between the nursing technician and the ophthalmologist. The Bland-Altman analysis showed a mean bias equal to zero for the VA measurements [95%-LoA: -0.25-0.25], 0.01 [95%-LoA: -0.86-0.88] for spherical equivalent (M), -0.08 [95%-LoA: -1.1-0.95] for keratometry (K) and -0.23 [95%-LoA: -4.4-4.00] for IOP. The measures had a high linear correlation (R [95%CI]: 0.87 [0.82-0.91]; 0.97 [0.96-0.98]; 0.96 [0.95-0.97] and 0.88 [0.84-0.91] respectively). The results observed demonstrate that remote ophthalmological data collection by adequately trained health professionals is viable. This confirms the utility and safety of these solutions for scenarios in which access to ophthalmologists is limited.

Ophthalmic image acquired by ophthalmologists and by allied health personnel as part of a telemedicine strategy: a comparative study of image quality.

OBJECTIVES: This study evaluates the quality of ophthalmic images acquired by a nurse technician trained in teleophthalmology as compared with images acquired by an ophthalmologist, in order to provide a better understanding of the workforce necessary to operate remote care programs. METHODS: A cross-sectional study was performed on 2044 images obtained from 118 participants of the TeleOftalmo project, in Brazil. Fundus and slit-lamp photography were performed on site by an ophthalmologist and by a nurse technician under the supervision of a remote ophthalmologist. Image quality was then evaluated by masked ophthalmologists. Proportion of suitable images in each group was compared. RESULTS: The proportion of concordant classification regarding quality was 94.8%, with a corrected kappa agreement of 0.94. When analyzing each type of photo separately, there was no significant difference in the proportion of suitable images between on-site ophthalmologist and nurse technician with remote ophthalmologist assistance for the following: slit-lamp views of the anterior segment and anterior chamber periphery, and fundus photographs centered on the macula and on the optic disc (P = 0.825, P = 0.997, P = 0.194, and P = 0.449, respectively). For slit-lamp views of the lens, the proportion of suitable images was higher among those obtained by an ophthalmologist (99.6%) than by a technician (93.8%, P < 0.01). CONCLUSIONS: Ophthalmic photographs acquired by a trained technician consistently achieved >90% adequacy for remote reading. Compared with ophthalmologist-acquired photos, the proportion of images deemed suitable achieved a high overall agreement. These findings provide favorable evidence of the adequacy of teleophthalmological imaging by nurse technicians.