Preparation of Biphenyl-Conjugated Bromotyrosine for Inhibition of PD-1/PD-L1 Immune Checkpoint Interactions.

Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 µM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 µM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.

Enhancement of Binding Affinity of Folate to Its Receptor by Peptide Conjugation.

(1) Background: The folate receptor (FR) is a target for cancer treatment and detection. Expression of the FR is restricted in normal cells but overexpressed in many types of tumors. Folate was conjugated with peptides for enhancing binding affinity to the FR. (2) Materials and Methods: For conjugation, folate was coupled with propargyl or dibenzocyclooctyne, and 4-azidophenylalanine was introduced in peptides for "click" reactions. We measured binding kinetics including the rate constants of association (ka) and dissociation (kd) of folate-peptide conjugates with purified FR by biolayer interferometry. After optimization of the conditions for the click reaction, we successfully conjugated folate with designed peptides. (3) Results: The binding affinity, indicated by the equilibrium dissociation constant (KD), of folate toward the FR was enhanced by peptide conjugation. The enhanced FR binding affinity by peptide conjugation is a result of an increase in the number of interaction sites. (4) Conclusion: Such peptide-ligand conjugates will be important in the design of ligands with higher affinity. These high affinity ligands can be useful for targeted drug delivery system.

Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and ß (hFRß).

Human folate receptors (hFRα and hFRß) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requires a large amount of hFRs. However, it is difficult to prepare active forms of hFRs from prokaryotic cells because of their high content of cysteine residues that form disulfide bonds. Here, we prepared active forms of hFRα and hFRß from inclusion bodies of Escherichia coli. The crucial steps in our preparation were intensive washing of the inclusion bodies to remove impurities derived from E. coli and gradual dropping of solubilized hFRs into refolding buffers to correctly reform disulfide bonds. The binding activity of prepared hFRs to folate was confirmed by biolayer interferometry measurements. Finally, we successfully prepared the active form of 2.52 mg hFRα and 2.4 mg hFRß from 10 g of E. coli cell bodies.

Biogenic Synthesis of Fluorescent Carbon Dots at Ambient Temperature Using Azadirachta indica (Neem) gum.

Synthesis of fluorescent Carbon Dots (CDs) from various carbonaceous materials apparently has acquired lots of interest amongst researchers as the corollary of the properties of CDs; which are subsequently getting unveiled. In this study we report the use of Azadirachta indica (Neem) Gum as a novel natural pre-cursor for synthesis of CDs at room temperature. Water soluble CDs of around 5-8 nm were obtained after treatment of the gum with ethanol and NaOH. These CDs exhibited green fluorescence in UV-light (λ = 365 nm). These CDs were found to be stable, having many bio-linkers attached on their surface, making it suitable for drug attachment and hence can serve as potential candidates for applications like drug delivery vehicles as well as for biosensors.

Biogenic gold nano-triangles: cargos for anticancer drug delivery.

We present synthesis of biogenic gold nano triangles (GNTs) using Azadirachta indica leaf extract at inherent pH (5.89) and its application in efficient drug delivery of doxorubicin (DOX) (anticancer drug). The main idea was to take advantage of large surface area of GNTs which has 3 dimensions and use the plant peptides coated on these triangles as natural linkers for the attachment of DOX. Sucrose density gradient centrifugation (SDGC) and dialysis methods were used for separation of the GNT from mixture of GNPs. Flocculation parameter (FP) was used to check stability of GNT which was found to be exceptionally high (0-0.75) due to the biological capping agents. DOX attachment to GNT was verified using Fourier transformed infra-red (FTIR) spectroscopy. The complex thus formed was found to be less toxic to normal cells (MDCK cells) and significantly toxic for the cancerous cells (HeLa cells). Drug loading efficiency was found to be 99.81% and DOX release followed first order release kinetics. Percentage drug release was found to be more than 4.5% in both acidic (5.8) as well as physiological pH (7.2) which is suitable for tumor targeting.