Sulfoglycodendron Antivirals with Scalable Architectures and Activities.

Many viruses initiate their cell-entry by binding their multisubunit receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multiprotein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed the detailed nature of binding between these antivirals and viral protein receptors and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.

Sulfoglycodendron Antivirals with Scalable Architectures and Activities.

Many viruses initiate their cell-entry by binding their multi-protein receptors to human heparan sulfate proteoglycans (HSPG) and other molecular components present on cellular membranes. These viral interactions could be blocked and the whole viruses could be eliminated by suitable HSPG-mimetics providing multivalent binding to viral protein receptors. Here, large sulfoglycodendron HSPG-mimetics of different topologies, structures, and sizes were designed to this purpose. Atomistic molecular dynamics simulations were used to examine the ability of these broad-spectrum antivirals to block multi-protein HSPG-receptors in HIV, SARS-CoV-2, HPV, and dengue viruses. To characterize the inhibitory potential of these mimetics, their binding to individual and multiple protein receptors was examined. In particular, vectorial distributions of binding energies between the mimetics and viral protein receptors were introduced and calculated along the simulated trajectories. Space-dependent residual analysis of the mimetic-receptor binding was also performed. This analysis revealed detail nature of binding between these antivirals and viral protein receptors, and provided evidence that large inhibitors with multivalent binding might act like a molecular glue initiating the self-assembly of protein receptors in enveloped viruses.

Correction to "Scalable Inhibitors of the Nsp3-Nsp4 Coupling in SARS-CoV-2".

[This corrects the article DOI: 10.1021/acsomega.2c06384.].

Antiviral fibrils of self-assembled peptides with tunable compositions.

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

Scalable Inhibitors of the Nsp3-Nsp4 Coupling in SARS-CoV-2.

The human Betacoronavirus SARS-CoV-2 is a novel pathogen claiming millions of lives and causing a global pandemic that has disrupted international healthcare systems, economies, and communities. The virus is fast mutating and presenting more infectious but less lethal versions. Currently, some small-molecule therapeutics have received FDA emergency use authorization for the treatment of COVID-19, including Lagevrio (molnupiravir) and Paxlovid (nirmaltrevir/ritonavir), which target the RNA-dependent RNA polymerase and the 3CLpro main protease, respectively. Proteins downstream in the viral replication process, specifically the nonstructural proteins (Nsps1-16), are potential drug targets due to their crucial functions. Of these Nsps, Nsp4 is a particularly promising drug target due to its involvement in the SARS-CoV viral replication and double-membrane vesicle formation (mediated via interaction with Nsp3). Given the degree of sequence conservation of these two Nsps across the Betacoronavirus clade, their protein-protein interactions and functions are likely to be conserved as well in SARS-CoV-2. Through AlphaFold2 and its recent advancements, protein structures were generated of Nsp3 and 4 lumenal loops of interest. Then, using a combination of molecular docking suites and an existing library of lead-like compounds, we virtually screened 7 million ligands to identify five putative ligand inhibitors of Nsp4, which could present an alternative pharmaceutical approach against SARS-CoV-2. These ligands exhibit promising lead-like properties (ideal molecular weight and log P profiles), maintain fixed-Nsp4-ligand complexes in molecular dynamics (MD) simulations, and tightly associate with Nsp4 via hydrophobic interactions. Additionally, alternative peptide inhibitors based on Nsp3 were designed and shown in MD simulations to provide a highly stable binding to the Nsp4 protein. Finally, these therapeutics were attached to dendrimer structures to promote their multivalent binding with Nsp4, especially its large flexible luminal loop (Nsp4LLL). The therapeutics tested in this study represent many different approaches for targeting large flexible protein structures, especially those localized to the ER. This study is the first work targeting the membrane rearrangement system of viruses and will serve as a potential avenue for treating viruses with similar replicative function.

Hierarchically Multivalent Peptide-Nanoparticle Architectures: A Systematic Approach to Engineer Surface Adhesion.

The multivalent binding effect has been the subject of extensive studies to modulate adhesion behaviors of various biological and engineered systems. However, precise control over the strong avidity-based binding remains a significant challenge. Here, a set of engineering strategies are developed and tested to systematically enhance the multivalent binding of peptides in a stepwise manner. Poly(amidoamine) (PAMAM) dendrimers are employed to increase local peptide densities on a substrate, resulting in hierarchically multivalent architectures (HMAs) that display multivalent dendrimer-peptide conjugates (DPCs) with various configurations. To control binding behaviors, effects of the three major components of the HMAs are investigated: i) poly(ethylene glycol) (PEG) linkers as spacers between conjugated peptides; ii) multiple peptides on the DPCs; and iii) various surface arrangements of HMAs (i.e., a mixture of DPCs each containing different peptides vs DPCs cofunctionalized with multiple peptides). The optimized HMA configuration enables significantly enhanced target cell binding with high selectivity compared to the control surfaces directly conjugated with peptides. The engineering approaches presented herein can be applied individually or in combination, providing guidelines for the effective utilization of biomolecular multivalent interactions using DPC-based HMAs.

Bimodal liquid biopsy for cancer immunotherapy based on peptide engineering and nanoscale analysis.

Despite its high potential, PD-L1 expressed by tumors has not been successfully utilized as a biomarker for estimating treatment responses to immunotherapy. Circulating tumor cells (CTCs) and tumor-derived exosomes that express PD-L1 can potentially be used as biomarkers; however, currently available assays lack clinically significant sensitivity and specificity. Here, a novel peptide-based capture surface is developed to effectively isolate PD-L1-expressing CTCs and exosomes from human blood. For the effective targeting of PD-L1, this study integrates peptide engineering strategies to enhance the binding strength and specificity of a ß-hairpin peptide derived from PD-1 (pPD-1). Specifically, this study examines the effect of poly(ethylene glycol) spacers, the secondary peptide structure, and modification of peptide sequences (e.g., removal of biologically redundant amino acid residues) on capture efficiency. The optimized pPD-1 configuration captures PD-L1-expressing tumor cells and tumor-derived exosomes with 1.5-fold (p = 0.016) and 1.2-fold (p = 0.037) higher efficiencies, respectively, than their whole antibody counterpart (aPD-L1). This enhanced efficiency is translated into more clinically significant detection of CTCs (1.9-fold increase; p = 0.035) and exosomes (1.5-fold increase; p = 0.047) from patients' baseline samples, demonstrating stronger correlation with patients' treatment responses. Additionally, we confirmed that the clinical accuracy of our system can be further improved by co-analyzing the two biomarkers (bimodal CTC/exosome analysis). These data demonstrate that pPD-1-based capture is a promising approach for capturing PD-L1-expressing CTCs and exosomes, which can be used as a reliable biomarker for cancer immunotherapy.

Factors Influencing Graft Endothelial Cell Density after Descemet Stripping Automated Endothelial Keratoplasty.

PURPOSE: To determine the factors that influence the endothelial cell density (ECD) of donor grafts after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This retrospective, interventional case series comprised 77 eyes of 64 patients who underwent DSAEK. Confocal microscopy was performed at the final follow-up examination to evaluate the endothelial cell count, cell morphology, and graft thickness. Univariate and multiple linear regression analyses were used to investigate recipient-, donor-, surgical-, and postoperative related variables capable of influencing graft endothelial cell counts after DSAEK. RESULTS: The mean patient age was 62.3 ± 15.6 years; patients were followed-up for 26.2 ± 20.9 months postoperatively. Forty-six eyes (59.7%) underwent stand-alone DSAEK; 31 eyes (40.3%) underwent DSAEK combined with cataract surgery. The donor trephination size was 8.0 ± 0.21 mm. The mean donor age was 30.4 ± 11.2 years, and the mean preoperative endothelial cell density was 3127.4 ± 315.1 cells/mm2, which decreased to 1788.6 ± 716.5 cells/mm2 postoperatively (P < 0.001). The mean postoperative central graft thickness was 102.4 ± 31.6 µm. Univariate analysis revealed that postoperative ECD was significantly associated with death to preservation time (P = 0.046), graft thickness (P = 0.016), follow-up duration (P = 0.005), and graft non-attachment (P = 0.049). Multiple regression analyses identified graft thickness (ß = 10.62, P = 0.003) and follow-up duration (ß = -22.09, P = 0.001) as the significant characteristics influencing postoperative ECD. CONCLUSION: The primary predictors of ECD after DSAEK were graft thickness and duration of follow-up. Surgeons' requests for ultrathin DSAEK donor grafts to improve visual outcomes might not have the desired postoperative outcome with respect to ECD.

Repeat Keratoplasty for Failed Deep Anterior Lamellar Keratoplasty in Keratoconus: Incidence, Indications, and Outcomes.

PURPOSE: To analyze the incidence, indications, and clinical outcomes of penetrating keratoplasty (PK) and repeat deep anterior lamellar keratoplasty (DALK) after failed DALK for keratoconus. METHODS: This retrospective interventional case series reviewed the medical records of patients with keratoconus who underwent DALK and were followed up for at least 1 year. Patients who underwent PK or repeat DALK for failed DALK were identified. The incidence, indications, and clinical outcomes after repeat keratoplasty were assessed. RESULTS: A total of 382 consecutive eyes with keratoconus underwent DALK over a 9-year period. Graft failure was observed in 14 eyes (3.7%). The reasons for the graft failure included nonhealing epithelial defects (n = 1), pseudoanterior chamber (n = 2), patient dissatisfaction with vision (n = 4), interface opacification (n = 4), high astigmatism (n = 1), and recurrence of keratoconus in the graft (n = 2). Of these 14 eyes with graft failure, 12 eyes underwent PK (n = 11) or repeat DALK (n = 1). At the most recent examination, which was performed 49.7 ± 25.2 months after the secondary graft, all regrafts were clear. There was no significant difference between eyes with clear first grafts and eyes that underwent PK or repeat DALK in visual outcomes at the final examination. Graft rejection was the most common complication, which was encountered in 75% of the regrafts. CONCLUSIONS: The prognosis for repeat keratoplasty was excellent, and the outcomes of secondary grafts were comparable to those of primary DALK grafts. However, the rate of rejection with secondary grafts was high, necessitating close follow-up after PK and repeat DALK performed for failed DALK.

Descemet Stripping Automated Endothelial Keratoplasty in Fuchs' Endothelial Dystrophy versus Pseudophakic Bullous Keratopathy.

PURPOSE: To compare clinical and confocal scan outcomes after Descemet stripping automated endothelial keratoplasty (DSAEK) performed for Fuchs' endothelial dystrophy (FED) versus pseudophakic bullous keratopathy (PBK). METHODS: This retrospective comparative study included 47 consecutive eyes of 39 patients with the diagnosis of FED (n = 29, group 1) or PBK (n = 18, group 2) that underwent DSAEK. Clinical outcomes were compared between the study groups. At the final follow-up examination, confocal microscopy was used to measure and compare central corneal and graft thickness as well as endothelial cell density and morphology between the two groups. RESULTS: Mean age at the time of surgery was 65.2 ± 11.8 and 69.4 ± 12.5 years in groups 1 and 2, respectively (P = 0.27). Follow-up period was 23.6 ± 14.0 months in group 1 and 25.6 ± 15.7 months in group 2 (P = 0.79). Postoperative best spectacle-corrected visual acuity was significantly better in group 1 than in group 2 until postoperative month 6. Afterwards, the two study groups were comparable in this regard. At the final follow-up examination, spherical equivalent refractive error was + 0.39 ± 1.46 diopters (D) in group 1 and + 0.80 ± 1.47 D in group 2 (P = 0.45). Postoperative keratometric astigmatism was 1.02 ± 0.83 D and 2.36 ± 0.67 D, respectively (P < 0.001). Mean central graft thickness was 98.0 ± 33.3 µm in group 1 and 107.6 ± 28.0 µm in group 2 (P = 0.45). No statistically significant difference was observed between the two groups in terms of the postoperative endothelial cell density. CONCLUSION: The outcomes of DSAEK surgery were comparable between FED and PBK. All grafts were clear despite the lower than normal endothelial cell counts.