Linker Length Research Articles

Design of Crosslinking Antibodies For T-Cell Activation: Experimental and Computational Analysis of PD-1/CD137 Bispecific Agents.

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VHdomains to determine the key factorsfor T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.

Self-assembly and functional activity of amphiphilic conjugates of the diterpenoid isosteviol and triphenylphosphonium cation, with focusing on biotechnological potential

Supramolecular systems based on amphiphilic triphenylphosphonium (TPP) conjugates of the diterpenoid isosteviol in which the diterpenoid skeleton and the TPP cation are linked by a polymethylene linker of varying length (n = 3, 6 or 8) have been fabricated. Elongation of this linker from 3 to 8 methylene units, allows for an 8-fold decrease in the amphiphile aggregation thresholds in aqueous solutions. These systems exhibit tunable self-assembly behavior, with the formation of both small aggregates (with a hydrodynamic diameter of ∼5–10 nm), as well as large vesicle-like structures (with a diameter of ∼50–100 nm), depending on the amphiphile concentration and length of the spacer fragment. The biotechnological potential of these amphiphiles has been demonstrated on the example of its membranotropic properties toward liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Significant capacity for integration into lipid bilayer has been revealed in these amphiphiles, which could be increased by extending the length of the polymethylene linker. Cationic liposome formulations have been prepared on the basis of these amphiphiles and DPPC by a noncovalent modification approach. Sustainability of its DH and zeta potential during longtime storage has been demonstrated. The studied systems exhibit a selective cytotoxic effect on M-HeLa cancer cell line and are a less toxic to normal Chang liver cells. The mechanism of the cytotoxic action of the liposomes is associated with the induction of apoptosis through mitochondrial pathway. The obtained formulations have successfully been applied for metronidazole encapsulation, characterized by high encapsulation efficiency and loading capacity, as well as prolonged time of drug release.

Fluorescence Labeling of Peptides: Finding the Optimal Protocol for Coupling Various Dyes to ATCUN-like Structures.

Labeling of peptides and proteins with fluorescent dyes is a key step in functionalizing these structures for a wide array of biological assays. However, coupling strategies of such dyes have not been optimized for the most common compounds, while this step is typically the most precious and costly of the whole synthesis. We searched for the best conditions for attachment of the most widely used fluorescent dyes such as 6-carboxyfluorescein, Rhodamine B, and BODIPY-FL to peptides, where amino terminal Cu(II) and Ni(II) binding site (ATCUN) peptides were used as a model system. Surprisingly, conventional methods of dye attachment proved to not be satisfactory and yielded poor efficiency results. We have discovered that when labeling primary amines on peptides, the uncommon synthesis of activated pentafluorophenol (PFP) esters is the most efficient strategy, expedited by microwave irradiation. Coupling to secondary amines is achieved most efficiently through conventional coupling reagents such as HATU and PyBOP. Furthermore, we have employed our fluorescently labeled ATCUN peptides in studies for Cu(II) and Ni(II) sensing, showing that changing the fluorophore does not significantly affect the fluorescence quenching process and discovering the optimal linker length between the ATCUN core and the dye, expanding the repertoire of fluorophores that can be used in this application.

Synthesis and Biological Evaluation of a Series of New Hybrid Amide Derivatives of Triazole and Thiazolidine-2,4-dione.

A series of hybrid compounds with triazole and thiazolidine nuclei connected by a linker has been synthesized and extensively studied. Various synthetic methods for the target compounds have been tested. A microbiological assessment of the obtained compounds was carried out on strains of pathogenic fungi C. albicans, C. non-albicans, multidrug-resistant C. auris, Rhizopus arrhizus, Aspergillus spp. and some dermatophytes and other yeasts. The lowest obtained MIC values for target compounds lie between 0.003 µg/mL and 0.5 µg/mL and therefore the compounds are not inferior or several times better than commercial azole drugs. The length of the acylpiperazine linker has a limited effect on antifungal activity. Some bioisosteric analogues were tested in microbiological analysis, but turned out to be weaker than the leader in activity. The highest activity was demonstrated by a compound with para-chlorobenzylidene substituent in the thiazolidine fragment. Molecular modelling was used to predict binding modes of synthesized molecules and rationalize experimentally observed SAR. The leader compound is twice more effective in inhibiting the formation of germ tubes by Candida albicans yeast cells compared to voriconazole. An increased level of Pdr5, an azoles drug efflux pump was observed, but the increase is lower than that caused by azoles. The results can be useful for further development of more powerful and safe antifungal agents.

Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation.

The development of agonists capable of activating the human complement system by binding to the C1 complex presents a novel approach for targeted cell killing. Bispecific nanobodies and Abs can successfully use C1 for this purpose; however, efficacy varies significantly between epitopes, Ab type, and bispecific design. To address this variability, we investigated monomeric agonists of C1 in the form of bispecific nanobodies, which lack Fc domains that lead to oligomerization in Abs. These therefore offer an ideal opportunity to explore the geometric parameters crucial for C1 activation. In this study, we explored the impact of linker length as a metric for Ag and epitope location. DNA nanotechnology and protein engineering allowed us to design linkers with controlled lengths and flexibilities, revealing a critical range of end-to-end distances for optimal complement activation. We discovered that differences in complement activation were not caused by differential C1 activation or subsequent cleavage of C4, but instead impacted C4b deposition and downstream membrane lysis. Considering the importance of Ab class and subclass, this study provides insights into the structural requirements of C1 binding and activation, highlighting linker and hinge engineering as a potential strategy to enhance potency over specific cellular targets. Additionally, using DNA nanotechnology to modify geometric parameters demonstrated the potential for synthetic biology in complement activation. Overall, this research offers valuable insights into the design and optimization of agonists for targeted cell killing through complement activation.

Zintl Clusters as a Platform for Lewis Acid Catalysis.

Clusters of the main group elements phosphorus and arsenic, commonly categorized as Zintl clusters, have been known for over a century. And, only now is the application of these systems as catalysts for organic synthesis being investigated. In this work, boranes are tethered via an aliphatic linker to Zintl-based clusters and their Lewis acidity is examined experimentally, by the Gutmann-Beckett test and competency in the hydroborative reduction of six organic substrates, as well as computationally, by fluoride ion affinity and hydride ion affinity methods. The effects of tuning the aliphatic linker length, substituents at the boron, and changing the cluster from a seven-atom phosphorus system to a seven-atom arsenic system on reactivity are studied.

Tethered Alkylammonium Dications as Electrochemical Interface Modifiers: Chain Length Effect on CO2 Reduction Selectivity at Industry-Relevant Current Density.

The electrochemical reduction of CO2 (CO2RR) has the potential to be an economically viable method to produce platform chemicals synergistically with renewable energy sources. Copper is one of the most commonly used electrocatalysts for this purpose, as it allows C-C bond formation, yielding a broad product distribution. Controlling selectivity is a stepping stone on the way to its industrial application. The kinetics of the reaction can be modified to favor the rates of certain products quickly and inexpensively by applying additives such as ionic liquids and coelectrolytes that directly affect the electrocatalytic interface. In this work, we propose tethered tetraalkylammonium salts as double-charged cationic modifiers of the electrochemical double layer to control CO2RR product selectivity. A novel setup comprising a gas diffusion electrode (GDE) flow cell coupled with real-time mass spectroscopy was used to study the effect of a library of the selected salts. We emphasize how the length of an alkyl linker effectively controls the selectivity of the reaction toward C1, C2, or C3 products at high relevant current densities (Jtotal = -400 mA cm-2) along with the inhibition of the parasitic hydrogen evolution reaction. Standard long-term experiments were performed for quantitative validation and stability evaluation. These results have broad implications for further tailoring an effective catalytic system for selective CO2 reduction reaction.

Albendazole oral drug delivery through ASBT-mediated cholic acid derivatives: A win-win for solubility and permeability

This study investigates the transmembrane transport of cholic acids through the primary sodium-dependent cholic acid transporter (ASBT) and explores the synthesis of a series of albendazole cholic acid derivatives (ABZ-CA). These derivatives, incorporating various linker lengths, aim to enhance the solubility and permeability of albendazole (ABZ) simultaneously.We systematically examined the structural and physicochemical properties of ABZ-CA, delving into its transmembrane transport mechanism and assessing its efficacy against Echinococcus multilocularis (E. multilocularis). Our investigation demonstrates the successful preparation of ABZ-CA using amino alcohols as linkers. The apparent solubility ranking of ABZ-CAs is as follows: ABZ-C4 > ABZ > ABZ-C8 ≈ ABZ-C1. This ranking reflects a balance between the solubilization effect induced by the amorphous state of ABZ-CA and the hydrophobic effect resulting from increased linker length.Enhanced absorption of ABZ-CAs is observed throughout all segments of the intestine, indicating a mechanism of passive diffusion. Notably, the optimal site for absorption shifts from the jejunum (ABZ) to the ileum (ABZ-CAs), with ASBT inhibitors significantly reducing the efficiency of transmembrane transport of ABZ-CAs in the ileum. This implies a specific mechanism of active transport reliant on ASBT. Our hypothesis is that ABZ-CAs undergo transmembrane transport in the gastrointestinal tract via a dual mechanism involving both passive diffusion and active transport.In pharmacodynamic studies, the inclusion of cholic acids does not affect the anti-E. multilocularis effect. Therefore, we believe that the formulation of ABZ-CAs not only enhances the solubility of ABZ but also increases its permeability, resulting in a synergistic effect. Moreover, this formulation presents a novel approach for enhancing the solubility and gastrointestinal permeability of BCSII and IV drugs during oral administration, consequently improving their oral bioavailability.

Amino Functionality Enables Aqueous Synthesis of Carboxylic Acid-Based MOFs at Room Temperature by Biomimetic Crystallization.

Enzyme immobilization within metal-organic frameworks (MOFs) is a promising solution to avoid denaturation and thereby utilize the desirable properties of enzymes outside of their native environments. The biomimetic mineralization strategy employs biomacromolecules as nucleation agents to promote the crystallization of MOFs in water at room temperature, thus overcoming pore size limitations presented by traditional postassembly encapsulation. Most biomimetic crystallization studies reported to date have employed zeolitic imidazole frameworks (ZIFs). Herein, we expand the library of MOFs suitable for biomimetic mineralization to include zinc(II) MOFs incorporating functionalized terephthalic acid linkers and study the catalytic performance of the enzyme@MOFs. Amine functionalization of terephthalic acids is shown to accelerate the formation of crystalline MOFs enabling new enzyme@MOFs to be synthesized. The structure and morphology of the enzyme@MOFs were characterized by PXRD, FTIR, and SEM-EDX, and the catalytic potential was evaluated. Increasing the linker length while retaining the amino moiety gave rise to a family of linkers; however, MOFs generated with the 2,2'-aminoterephthalic acid linker displayed the best catalytic performance. Our data also illustrate that the pH of the reaction mixture affects the crystal structure of the MOF and that this structural transformation impacts the catalytic performance of the enzyme@MOF.

Development of a Stable Peptide-Major Histocompatibility Complex (MHC) via Sortase and Click Chemistry.

T cells play a crucial role in antitumor immune responses and the clearance of infected cells. They identify their targets through the binding of T-cell receptors (TCRs) to peptide-major histocompatibility complex (pMHC) molecules present in cancer cells, infected cells, and antigen-presenting cells. This interaction is often weak, requiring multimeric pMHC molecules to enhance the avidity for identifying antigen-specific T cells. Current exchangeable pMHC-I tetramerization methods may overlook TCRs recognizing less stable yet immunogenic peptides. In vivo applications targeting antigen-specific T cells demand the genetic synthesis of a pMHC fusion for each unique peptide antigen, which poses a significant challenge. To address these challenges, we developed a sortase and click chemistry-mediated approach for generating stable pMHC molecules. Leveraging sortase technology, we introduced an azide click-handle near the N-terminus of β2m, proximal to the MHC-peptide-binding groove. Simultaneously, the peptide was engineered with a multi glycine linker and a C-terminal alkyne click-handle. Azide-alkyne click reactions efficiently immobilized the peptide onto the MHC molecule, providing a versatile and efficient method for pMHC generation. The resulting peptide-clicked-MHC specifically binds to its cognate TCR and remains stable for over 3 months at 4 °C in the absence of any additional free peptide. The stability of the pMHC and its affinity to cognate TCRs are influenced by the linker's nature and length. Multi glycine linkers outperform poly(ethylene glycol) (PEG) linkers in this regard. This technology expands the toolkit for identifying and targeting antigen-specific T cells, enhancing our understanding of cancer-specific immune responses, and has the potential to streamline the development of personalized immunotherapies.

A Systematic Study of Nonionic Di- and Multiborane Catalysts for the Oligomerization and Polymerization of Epoxides.

Borane catalysis has emerged as a powerful technology in epoxide polymerization. Still, the structure-activity correlations for these catalysts are not fully understood to date, especially regarding compounds with nonionic backbones. Thus, in this work, 13 different borane catalysts of this respective type are described and investigated for their epoxide oligomerization and polymerization performance, using propylene oxide (PO), 1-butylene oxide (BO) and allyl glycidyl ether (AGE) as monomers. Structurally, special emphasis is put on catalysts with different linker lengths and linker flexibilities as well as the introduction of more than two borane functionalities. Importantly, this screening is conducted both under typical polymerization conditions as well as under the chain transfer agent (CTA)-rich conditions relevant for large-scale production. It is found that suitable preorganization of the borane groups, such as present in biphenyl derivatives, offers a simple route to high-performing catalysts and quantitative monomer conversion of the investigated epoxides. Furthermore, it is demonstrated that a diborane-catalyzed oligomerization can be kept active over weeks, whereby repeated addition of monomer batches (14 steps) constantly results in full conversion and well-defined oligoethers, underlining the practical potential of this method. The absence of co-initiating counter ions is suggested as an inherent advantage of nonionic catalysts.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests functionalizing DCA with a headgroup (e.g., phosphatidylcholine, PC) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC headgroup chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC headgroup enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC headgroup altered the extrahepatic accumulation of siRNA, with optimal linker length being different for different tissues. Increasing PC headgroup valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel phosphocholine linker variants for chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for extrahepatic distribution of siRNAs.

Chelator boosted tumor-retention and pharmacokinetic properties: development of 64Cu labeled radiopharmaceuticals targeting neurotensin receptor.

Accumulating evidence suggests that neurotensin (NTS) and neurotensin receptors (NTSRs) play key roles in lung cancer progression by triggering multiple oncogenic signaling pathways. This study aims to develop Cu-labeled neurotensin receptor 1 (NTSR1)-targeting agents with the potential for both imaging and therapeutic applications. A series of neurotensin receptor antagonists (NRAs) with variable propylamine (PA) linker length and different chelators were synthesized, including [64Cu]Cu-CB-TE2A-iPA-NRA ([64Cu]Cu-4a-c, i = 1, 2, 3), [64Cu]Cu-NOTA-2PA-NRA ([64Cu]Cu-4d), [64Cu]Cu-DOTA-2PA-NRA ([64Cu]Cu-4e, also known as [64Cu]Cu-3BP-227), and [64Cu]Cu-DOTA-VS-2PA-NRA ([64Cu]Cu-4f). The series of small animal PET/CT were conducted in H1299 lung cancer model. The expression profile of NTSR1 was also confirmed by IHC using patient tissue samples. For most of the compounds studied, PET/CT showed prominent tumor uptake and high tumor-to-background contrast, but the tumor retention was strongly influenced by the chelators used. For previously reported 4e, [64Cu]Cu-labeled derivative showed initial high tumor uptake accompanied by rapid tumor washout at 24h. The newly developed [64Cu]Cu-4d and [64Cu]Cu-4f demonstrated good tumor uptake and tumor-to-background contrast at early time points, but were less promising in tumor retention. In contrast, our lead compound [64Cu]Cu-4b demonstrated 9.57 ± 1.35, 9.44 ± 2.38 and 9.72 ± 4.89%ID/g tumor uptake at 4, 24, and 48h p.i., respectively. Moderate liver uptake (11.97 ± 3.85, 9.80 ± 3.63, and 7.72 ± 4.68%ID/g at 4, 24, and 48h p.i.) was observed with low uptake in most other organs. The PA linker was found to have a significant effect on drug distribution. Compared to [64Cu]Cu-4b, [64Cu]Cu-4a had a lower background, including a greatly reduced liver uptake, while the tumor uptake was only moderately reduced. Meanwhile, [64Cu]Cu-4c showed increased uptake in both the tumor and the liver. The clinical relevance of NTSR1 was also demonstrated by the elevated tumor expression in patient tissue samples. Through the side-by-side comparison, [64Cu]Cu-4b was identified as the lead agent for further evaluation based on its high and sustained tumor uptake and moderate liver uptake. It can not only be used to efficiently detect NTSR1 expression in lung cancer (for diagnosis, patient screening, and treatment monitoring), but also has the great potential to treat NTSR-positive lesions once chelating to the beta emitter 67Cu.

Phenyl-Substituted Cage Silsesquioxane-Based Star-Shaped Giant Molecular Clusters: Synthesis, Properties, and Surface Segregation Behavior.

The crystallinity, solubility, and physical properties of polyhedral oligomeric silsesquioxane (POSS) compounds are highly dependent on their organic substituents. We previously synthesized a series of isobutyl-substituted star-shaped POSS derivatives with aliphatic chain linkers of different length. In this study, we prepared C3- and C6-linked phenyl-substituted star-shaped POSS derivatives (3Ph-C3 and 3Ph-C6) by the hydrosilylation of heptaphenylallyl- and hexenyl-POSS (1a and 1b) and octadimethylsiloxy-Q8-silsesquioxane (Q8M8H) (2), respectively, and their properties were compared with those of the corresponding isobutyl-substituted derivatives (5iBu-C3 and 5iBu-C6). Although 3Ph-C6 was only soluble in chloroform and insoluble in tetrahydrofuran (THF) and toluene, 3Ph-C3 was soluble even in THF and toluene, suggesting that the shorter linkers of the derivative afford a wider range of solvents for dissolution. Differential scanning calorimetry analysis showed that 3Ph-C3 exhibited a baseline shift at 190 °C and an endothermic peak at 316 °C. However, no clear baseline shift was observed for 3Ph-C6. Thermogravimetric analysis showed that the shorter linker in the phenyl-substituted star-shaped POSS derivative significantly increased the decomposition temperature compared with the longer linker. The annealed cast film of 3Ph-C3 at 340 °C above its melting temperature formed a transparent film even after cooling to room temperature. However, an opaque whitish film was formed in the case of 3Ph-C6. Poly(methyl methacrylate) (PMMA) films containing 2 wt % 3Ph-C3 and 3Ph-C6 were prepared by casting their chloroform solutions onto glass substrates overnight at room temperature. The static water contact angle measurements and XPS analysis for the castings film containing 3Ph-C3 and 3Ph-C6 suggested that degree of the segregation amount of 3Ph-C3 was larger than that of 3Ph-C6. The shorter linker length in the phenyl-substituted star-shaped POSS derivative, 3Ph-C3, with its greater predicted solubility in PMMA, exhibited entropy-driven surface segregation.

Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA.

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

2D and 3D anticancer properties of C2-functionalised glucosamine-Pt (IV) prodrugs based on cisplatin scaffold.

A series of C2-functionalied Pt (IV) glycoconjugates based on glucosamine have been synthesised, characterised and tested as anticancer agents on a series of different 2D and 3D cancer cell lines. The carbohydrate will act as a targeted delivery system to improve the selectivity, exploiting the Warburg Effect and the GLUTs receptors that are overexpressed in most of the cancer cells. The hydroxyl at C2 of the carbohydrates does not participate in hydrogen bonding with the GLUTs receptors, making C2 an attractive position for drug conjugation as seen in literature. In this study, we use the amino functionality at the C2 position in glucosamine and Copper-catalysed Azide-Alkyne Cycloaddition "click" (CuAAC) reaction to connect the prodrug Pt (IV) scaffold to the carbohydrate. We have investigated complexes with different linker lengths, as well as acetyl protected and free derivatives. To the best of our knowledge, this study represents the first series of Pt (IV) glucosamine-conjugates functionalised at C2.

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60–90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid.

Photo-regulated PROTACs: A novel tool for temporal control of targeted protein degradation

PROTACs (Proteolysis targeting chimeras) are chimeric molecules designed to induce targeted protein degradation via the ubiquitin–proteasome system. These molecules catalytically degrade target proteins and sustainably inhibit their function. Therefore, PROTAC's unique mechanism of action is not only beneficial in medicine but also serves as a valuable tool for molecular biological analysis in fields like chemical biology, biochemistry, and drug discovery. This study presents a novel turn-off (ON-OFF) type PROTAC development strategy utilizing a photocleavable linker. The inclusion of this linker enables temporal control of the degradation activity targeting BRD4 protein upon UV light exposure. PROTAC-2 demonstrated the most potent degradation activity against BRD4 among the other synthesized PROTACs with varying linker lengths. The UV light-induced cleavage of PROTAC-2 was confirmed, leading to a reduction in its BRD4 degradation activity. Notably, this study introduces a novel linker capable of nullifying degradation activity of PROTACs which is activated by light irradiation. These findings offer a promising strategy for the development of turn-off type PROTACs, providing enhanced temporal control over protein degradation. The approach holds significant potential for applications in molecular function studies and drug discovery.

Analogues of the pan-selectin antagonist rivipansel (GMI-1070)

The selectin family consisting of E−, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E−, as well as P-selectin.

Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC50 < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.