Cyclic ß2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

Synthesis and Structural Characterization of Macrocyclic α-ABpeptoids and Their DNA-Encoded Library.

The first synthesis of macrocyclic α-ABpeptoids with varying lengths is described. X-ray crystal structures reveal that cyclic trimer displays a chair-like conformation with a cct amide sequence and cyclic tetramer has a saddle-like structure with an uncommon cccc amide arrangement. The creation of a DNA-encoded combinatorial library of macrocyclic α-ABpeptoids is described.

Hemostatic efficacy of a flowable collagen-thrombin matrix during coronary artery bypass grafting: a double-blind randomized controlled trial.

BACKGROUND: Flowable hemostatic agents have the advantage of being able to be applied to irregular wound surfaces and difficult to reach areas. We sought to compare the effectiveness and safety of the flowable hemostatic sealants Collastat® (collagen hemostatic matrix, [CHM]) and Floseal® (gelatin hemostatic matrix, [GHM]) during off-pump coronary artery bypass (OPCAB). METHODS: In this prospective, double-blind, randomized controlled trial, 160 patients undergoing elective OPCAB surgery were enrolled between March 2018 and February 2020. After primary suture of the aortocoronary anastomosis, an area of hemorrhage was identified, and patients received either CHM or GHM (n = 80, each). Study endpoints were the following: proportion of successful intraoperative hemostasis and time required for hemostasis overall postoperative bleeding, proportion of transfusion of blood products, and surgical revision for bleeding. RESULTS: Of the total patients, 23% were female, and the mean age was 63 years (range 42-81 years). Successful hemostasis proportion within 5 min was achieved for 78 patients (97.5%) in the GHM group, compared to 80 patients (100%) in the CHM group (non-inferiority p = 0.006). Two patients receiving GHM required surgical revision to achieve hemostasis. There were no differences in the mean time required to obtain hemostasis [GHM vs. CHM, mean 1.49 (SD 0.94) vs. 1.35 (0.60) min, p = 0.272], as confirmed by time-to-event analysis (p = 0.605). The two groups had similar amounts of mediastinal drainage for 24 h postoperatively [538.5 (229.1) vs. 494.7 (190.0) ml, p = 0.298]. The CHM group required less packed red blood cells, fresh frozen plasma, and platelets for transfusion than the GHM group (0.5 vs. 0.7 units per patient, p = 0.047; 17.5% vs. 25.0%, p = 0.034; 7.5% vs. 15.0%, p = 0.032; respectively). CONCLUSIONS: CHM was associated with a lower need for FFP and platelet transfusions. Thus, CHM is a safe and effective alternative to GHM. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04310150.

Fractional centralities on networks: Consolidating the local and the global.

We propose a new centrality incorporating two classical node-level centralities, the degree centrality and the information centrality, which are considered as local and global centralities, respectively. These two centralities have expressions in terms of the graph Laplacian L, which motivates us to exploit its fractional analog L^{γ} with a fractional parameter γ. As γ varies from 0 to 1, the proposed fractional version of the information centrality makes intriguing changes in the node centrality rankings. These changes could not be generated by the fractional degree centrality since it is mostly influenced by the local aspect. We prove that these two fractional centralities behave similarly when γ is close to 0. This result provides its complete understanding of the boundary of the interval in which γ lies since the fractional information centrality with γ=1 is the usual information centrality. Moreover, our computation for the correlation coefficients between the fractional information centrality and the degree centrality reveals that the fractional information centrality is transformed from a local centrality into being a global one as γ changes from 0 to 1.

Evaluation of the cell permeability of bicyclic peptoids and bicyclic peptide-peptoid hybrids.

Bicyclization has proven to be an effective strategy for significantly restricting conformational flexibility in peptides and peptidomimetics such as peptoids. Such constrained bicyclic peptoids would have far higher conformational rigidity than monocyclic and linear ones, allowing them to have enhanced binding affinity and selectivity for their biological targets. Herein, we show that bicyclic peptoids have superior cellular uptake efficiency than their linear counterparts regardless of their side chains and ring sizes. As a representative example, an 8-mer bicyclic peptoid achieves a CP50 value of 1.2 µM, which is > 5-times superior to the corresponding linear peptoid. Additionally, we also demonstrate that bicyclic peptide-peptoid hybrids are much more cell-permeable than native peptides. Due to their favorable properties including improved cellular uptake, resistance to proteolytic degradation, relatively large sizes, and enormous structural diversity, constrained bicyclic peptoids and peptide-peptoid hybrids will play an important role as potential drug leads, especially in targeting intracellular protein-protein interactions, which are traditionally considered undruggable.

Design and synthesis of a DNA-encoded combinatorial library of bicyclic peptoids.

Here we describe the design and synthesis of a DNA-encoded library of bicyclic peptoids. We show that our solid-phase strategy is facile and DNA-compatible, yielding a structurally diverse combinatorial library of bicyclic peptoids of various ring sizes. We also demonstrate that affinity-based screening of a DNA-encoded library of bicyclic peptoids enables to efficiently identify high-affinity ligands for a target protein. Given their highly constraint structures, as well as increased cell permeability and proteolytic stability relative to native peptides, bicyclic peptoids could be an excellent source of protein capture agents. As such, our DNA-encoded library of bicyclic peptoids will serve as versatile tools that facilitate the generation of potent ligands against many challenging targets, such as intracellular protein-protein interactions.

Thrombolytic Agents: Nanocarriers in Controlled Release.

Thrombosis is a life-threatening pathological condition in which blood clots form in blood vessels, obstructing or interfering with blood flow. Thrombolytic agents (TAs) are enzymes that can catalyze the conversion of plasminogen to plasmin to dissolve blood clots. The plasmin formed by TAs breaks down fibrin clots into soluble fibrin that finally dissolves thrombi. Several TAs have been developed to treat various thromboembolic diseases, such as pulmonary embolisms, acute myocardial infarction, deep vein thrombosis, and extensive coronary emboli. However, systemic TA administration can trigger non-specific activation that can increase the incidence of bleeding. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses. To overcome these limitations, various types of nanocarriers have been introduced that enhance the pharmacokinetic effects by protecting the TA from the biological environment and targeting the release into coagulation. The nanocarriers show increasing half-life, reducing side effects, and improving overall TA efficacy. In this work, the recent advances in various types of TAs and nanocarriers are thoroughly reviewed. Various types of nanocarriers, including lipid-based, polymer-based, and metal-based nanoparticles are described, for the targeted delivery of TAs. This work also provides insights into issues related to the future of TA development and successful clinical translation.

DNA-Encoded Combinatorial Library of Macrocyclic Peptoids.

We report the design and synthesis of a DNA-encoded one-bead one-compound library of cyclic peptoids composed of more than 11 million molecules. We show that affinity-based screening of this large library can identify cyclic peptoid ligands for a target protein. In this work, we developed a simple method for amplifying the PCR product from DNA tags on a single bead, thereby enabling determination of the structures of hit cyclic peptoids with no need for high-throughput sequencing and complicated data analysis. We also developed a sublibrary screening strategy to minimize false positives caused by the interference of coding DNA tags before starting laborious and impractical hit confirmation. Given the simplicity and robustness of the synthesis and screening, along with the desirable features of macrocyclic peptoids including improved conformational rigidity, our method will be highly useful for discovering biologically active molecules modulating challenging targets such as protein-protein interactions that are not easily targeted by typical peptidomimetics and small-molecules.

Oligomers of α-ABpeptoid/ß3 -peptide hybrid.

Peptoids, oligomers of N-substituted glycines, have been attracting increasing interest due to their advantageous properties as peptidomimetics. However, due to the lack of chiral centers and amide hydrogen atoms, peptoids, in general, do not form folding structures except that they have α-chiral side chains. We have recently developed "peptoids with backbone chirality" as a new class of peptoid foldamers called α-ABpeptoids and demonstrated that they could have folding conformations owing to the methyl groups on chiral α-carbons in the backbone structure. Here we report α-ABpeptoid/ß3 -peptide oligomers as a unique peptidomimetic structure with a heterogeneous backbone. This hybrid structure contains a mixed α-ABpeptoid and ß3 -peptide residues arranged in an alternate manner. These α-ABpeptoid/ß3 -peptide oligomers could form intramolecular hydrogen bonding and have better cell permeability relative to pure peptide sequences. These oligomers were shown to adopt ordered folding structures based on circular dichroism studies. Overall, α-ABpeptoid/ß3 -peptide oligomers may represent a novel class of peptidomimetic foldamers and will find a wide range of applications in biomedical and material sciences.

Solid-Phase Synthesis and Circular Dichroism Study of ß-ABpeptoids.

The development of peptidomimetic foldamers that can form well-defined folded structures is highly desirable yet challenging. We previously reported on α-ABpeptoids, oligomers of N-alkylated ß²-homoalanines and found that due to the presence of chiral methyl groups at α-positions, α-ABpeptoids were shown to adopt folding conformations. Here, we report ß-ABpeptoids having chiral methyl group at ß-positions rather than α-positions as a different class of peptoids with backbone chirality. We developed a facile solid-phase synthetic route that enables the synthesis of ß-ABpeptoid oligomers ranging from 2-mer to 8-mer in excellent yields. These oligomers were shown to adopt ordered folding conformations based on circular dichroism (CD) and NMR studies. Overall, these results suggest that ß-ABpeptoids represent a novel class of peptidomimetic foldamers that will find a wide range of applications in biomedical and material sciences.

Submonomer Strategy toward Divergent Solid-Phase Synthesis of α-ABpeptoids.

A novel submonomer solid-phase synthetic method for α-ABpeptoid oligomers is reported. Iterative submonomer coupling and Fukuyama-Mitsunobu alkylation enable facile, divergent synthesis of α-ABpeptoid oligomers substituted with chemically diverse side chains in excellent yields.

Transfer-molded wrappable microneedle meshes for perivascular drug delivery.

After surgical procedures such as coronary/peripheral bypass grafting or endarterectomy for the treatment of organ ischemia derived from atherosclerosis, intimal hyperplasia (IH) which leads to restenosis or occlusion at the site of graft anastomosis frequently occurs. In order to inhibit IH caused by abnormal growth of smooth muscle cells (SMCs) in tunica media, various perivascular drug delivery devices are reported for delivery of anti-proliferation drugs into vascular tissue. However, there still remain conflicting requirements such as local and unidirectional delivery vs device porosity, and conformal tight device installation vs pulsatile expansion and constriction of blood vessels. In this study, a biodegradable microneedle (MN) array is developed on a flexible woven surgical mesh using a transfer molding method. Mechanical properties of 'wrappable' MN meshes are investigated and compared to the properties of blood vessels. Ex vivo and in vivo animal studies demonstrate enhanced drug delivery efficiency, efficacy for IH reduction, and safety of MN mesh. In particular, MN mesh showed significantly reduced neointiamal formation (11.1%) compared to other competitive groups (23.7 and 22.2%) after 4-week in vivo animal study. Additionally, wrappable MN meshes effectively suppressed side effects such as IH due to mechanical constriction, loss of toxic drug to the surroundings, and cell death that were frequently observed with other previous perivascular drug delivery devices.

A Biodegradable Microneedle Cuff for Comparison of Drug Effects through Perivascular Delivery to Balloon-Injured Arteries.

Restenosis at a vascular anastomosis site is a major cause of graft failure and is difficult to prevent by conventional treatment. Perivascular drug delivery has advantages as drugs can be diffused to tunica media and subintima while minimizing the direct effect on endothelium. This in vivo study investigated the comparative effectiveness of paclitaxel, sirolimus, and sunitinib using a perivascular biodegradable microneedle cuff. A total of 31 New Zealand white rabbits were used. Rhodamine was used to visualize drug distribution (n = 3). Sirolimus- (n = 7), sunitinib- (n = 7), and paclitaxel-loaded (n = 7) microneedle cuffs were placed at balloon-injured abdominal aortae and compared to drug-free cuffs (n = 7). Basic histological structures were not affected by microneedle devices, and vascular wall thickness of the device-only group was similar to that of normal artery. Quantitative analysis revealed significantly decreased neointima formation in all drug-treated groups (p < 0.001). However, the tunica media layer of the paclitaxel-treated group was significantly thinner than that of other groups and also showed the highest apoptotic ratio (p < 0.001). Proliferating cell nuclear antigen (PCNA)-positive cells were significantly reduced in all drug-treated groups. Sirolimus or sunitinib appeared to be more appropriate for microneedle devices capable of slow drug release because vascular wall thickness was minimally affected.

Microneedle-based minimally-invasive measurement of puncture resistance and fracture toughness of sclera.

UNLABELLED: The sclera provides the structural support of the eye and protects the intraocular contents. Since it covers a large portion of the eye surface and has relatively high permeability for most drugs, the sclera has been used as a major pathway for drug administration. Recently, microneedle (MN) technology has shown the possibility of highly local and minimally-invasive drug delivery to the eye by MN insertion through the sclera or the suprachoroidal space. Although ocular MN needs to be inserted through the sclera, there has been no systematic study to understand the mechanical properties of the sclera, which are important to design ocular MNs. In this study, we investigated a MN-based method to measure the puncture resistance and fracture toughness of the sclera. To reflect the conditions of MN insertion into the sclera, force-displacement curves obtained from MN-insertion tests were used to estimate the puncture resistance and fracture toughness of sclera tissue. To understand the effect of the insertion conditions, dependency of the mechanical properties on insertion speeds, pre-strain of the sclera, and MN sizes were analyzed and discussed. STATEMENT OF SIGNIFICANCE: Measurement of mechanical property of soft biological tissue is challenging due to variations between tissue samples or lack of well-defined measurement techniques. Although non-invasive measurement techniques such as nano/micro indentation were employed to locally measure the elastic modulus of soft biological materials, mechanical properties such as puncture resistance or fracture toughness, which requires "invasive" measurement and is important for the application of "microneedles or hypodermic needles", has not been well studied. In this work, we report minimally-invasive measurement of puncture resistance and fracture toughness of sclera using a double MN insertion method. Parametric studies showed that use of MN proved to be advantageous because of minimally-invasive insertion into tissue as well as higher sensitivity to sub-tissue architecture during the measurement.

Oligomers of N-Substituted ß(2)-Homoalanines: Peptoids with Backbone Chirality.

A new class of peptoid-based peptidomimetics composed of oligomers of N-substituted ß(2)-homoalanines is reported. Design, solid-phase synthesis, and preliminary circular dichroism studies of oligomers of N-alkylated ß(2)-homoalanines consisting of up to 8-mers are described.

Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery.

It has been challenging for microneedles to deliver drugs effectively to thin tissues with little background support such as the cornea. Herein, we designed a microneedle pen system, a single microneedle with a spring-loaded microneedle applicator to provide impact insertion. To firmly attach solid microneedles with 140 µm in height at the end of macro-scale applicators, a transfer molding process was employed. The fabricated microneedle pens were then applied to mouse corneas. The microneedle pens successfully delivered rhodamine dye deep enough to reach the stromal layer of the cornea with small entry only about 1000 µm(2). When compared with syringes or 30 G needle tips, microneedle pens could achieve more localized and minimally invasive delivery without any chances of perforation. To investigate the efficacy of microneedle pens as a way of drug delivery, sunitinib malate proven to inhibit in vitro angiogenesis, was delivered to suture-induced angiogenesis model. When compared with delivery by a 30 G needle tip dipped with sunitinib malate, only delivery by microneedle pens could effectively inhibit corneal neovascularization in vivo. Microneedle pens could effectively deliver drugs to thin tissues without impairing merits of using microneedles: localized and minimally invasive delivery.

Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds.

Thermal drawing is a versatile rapid prototyping method that can freely form microneedle (MN) structures with ultra-high aspect ratio without relying on any complex and expensive process. However, it is still challenging to repeatedly produce MNs with identical shapes using this thermal drawing due to small fluctuations in processing conditions such as temperatures, drawing speeds, drawing heights, or parallelism in the drawing setup. In addition, thermal drawing is only applicable to thermoplastic materials and most natural biomaterials are incompatible with this method. Thus, we propose use of thermal drawing to fabricate master molds with high aspect ratios and replicate the shape by micromolding. In this work, high A/R MNs with various body profiles were fabricated by thermal drawing and replicated to silk fibroin (SF) MNs multiple times using micromolding. The original MN shape was precisely copied to the SF MNs. Methanol treatment enhanced the mechanical strength of SF MNs up to about 113% more depending on the treatment duration. We also demonstrated that methanol exposure time could effectively control drug release rates from SF MNs.

Facile method to sequence cyclic peptides/peptoids via one-pot ring-opening/cleavage reaction.

A facile method for sequence determination of cyclic peptides/peptoids is described. Macrocyclic peptides/peptoids of 3-10 residues were efficiently synthesized through thioether formation. One-pot reaction of thioether-embedded cyclic peptides/peptoids involving cyanogen bromide-mediated ring-opening and cleavage provides linearized molecules, which can be efficiently sequenced by tandem mass spectrometry.

Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury.

Restenosis often occurs at the site of vascular grafting and may become fatal for patients. Restenosis at anastomosis sites is due to neointimal hyperplasia (NH) and difficult to treat with conventional treatments. Such abnormal growth of smooth muscle cells in tunica media of vascular tissue can be reduced by delivering anti-proliferation drugs such as paclitaxel (PTX) to the inner vascular layer. Drug eluting stents (DES) or drug eluting balloon (DEB) have been developed to treat such vascular diseases. However, they are less efficient in drug delivery due to the drug loss to blood stream and inadequate to be applied to re-stenotic area in the presence of stent or anastomosis sites. Recently, we have introduced microneedle cuff (MNC) as perivascular delivery devices to achieve high delivery efficiency to tunica media. In this study, we investigated in vivo microneedle insertion and efficacy in treating NH using a rabbit balloon injury model. Microneedle shape was optimized for reliable insertion into tunica media layer. Uniform distribution of PTX in tunica media delivered by MNC devices was also confirmed. Animal study demonstrated significant NH reduction by MNC treatments and much higher delivery efficiency than flat type devices.