A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system.

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli-based in vitro coupled transcription-translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

Cell-free protein synthesis of CD1E and B2M protein and in vitro interaction.

CD1E, one of the most important glycolipid antigens on T cell membranes, is required for glycolipid antigen presentation on the cell surface. Cell-based recombinant expression systems have many limitations for synthesizing transmembrane proteins such as CD1E, including low protein yields and miss folding. To overcome these challenges, here we successfully synthesized high-quality soluble CD1E using an E.coli cell-free protein synthesis system (CFPS) with the aid of detergent. Following purification by Ni2+ affinity chromatography, we were able to obtain CD1E with ≥90% purity. Furthermore, we used the string website to predict the protein interaction network of CD1E and identified a potential binding partner━B2M. Similarly, we synthesized soluble B2M in the E.coli CFPS. Finally, we verified the interaction between CD1E and B2M by using Surface Plasmon Resonance (SPR). Taken together, the methods described here provide an alternative way to obtain active transmembrane protein and may facilitate future structural and functional studies on CD1E.

Transition Metal Catalyst-Free C-3 Sulfonylmethylation of Imidazo[1,2-a]pyridines with Glyoxylic Acid and Sodium Sulfinates in Water.

Herein, we describe an efficient and benign protocol for direct C-3 sulfonylmethylation of imidazo[1,2-a]pyridines with glyoxylic acid and sodium sulfinates. Various sulfonylmethylated imidazo[1,2-a]pyridines were synthesized in water under transition metal catalyst-free conditions. This multicomponent reaction featured available substrates, good functional group tolerance, moderate to excellent yields, and mild reaction conditions.

Copper-Catalyzed C-3 Functionalization of Imidazo[1,2-a]pyridines with 3-Indoleacetic Acids.

A copper-catalyzed C-3 functionalization of imidazo[1,2-a]pyridines with 3-indoleacetic acids through an aerobic oxidative decarboxylative process has been developed. The protocol provided a series of 3-(1H-indol-3-ylmethyl)-imidazo[1,2-a]pyridines in moderate to good yields under simple reaction conditions. Importantly, some products exhibited potent antiproliferative activity in cancer cell lines.

Discovery of KRas G12C-IN-3 and Pomalidomide-based PROTACs as degraders of endogenous KRAS G12C with potent anticancer activity.

A series of KRAS G12C-targeting PROTACs (PROteolysis TArgeting Chimeras) were designed and synthesized based on KRas G12C-IN-3 (a KRAS G12C inhibitor) and pomalidomide as degraders of KRAS G12C with a molecular weight of < 900. Among them, compound KP-14 (m.w. = 852.16; tPSA = 174.53) showed the highest KRAS G12C-degrading capability in NCI-H358 cancer cells (DC50≈1.25 µM). KP-14 bound to KRAS G12C through the acrylamide warhead and recruited the E3 ligase CRBN, causing rapid and sustained KRAS G12C degradation which led to suppression of MAPK signaling pathway in NCI-H358 cells. In addition, KP-14 selectively induced the degradation of KRAS G12C but not other KRAS isoforms such as G13D via PROTAC mechanism. Furthermore, KP-14 exhibited potent antiproliferative activity against NCI-H358 cancer cells and was able to suppress the formation of NCI-H358 tumor colonies. Collectively, this work suggests that KP-14 may serve as a tool compound for exploring the degradation of KRAS G12C by PROTAC strategy and deserve further investigation as a potential anticancer agent.

Small molecule-based immunomodulators for cancer therapy.

Immunotherapy has led to a paradigm shift in the treatment of cancer. Current cancer immunotherapies are mostly antibody-based, thus possessing advantages in regard to pharmacodynamics (e.g., specificity and efficacy). However, they have limitations in terms of pharmacokinetics including long half-lives, poor tissue/tumor penetration, and little/no oral bioavailability. In addition, therapeutic antibodies are immunogenic, thus may cause unwanted adverse effects. Therefore, researchers have shifted their efforts towards the development of small molecule-based cancer immunotherapy, as small molecules may overcome the above disadvantages associated with antibodies. Further, small molecule-based immunomodulators and therapeutic antibodies are complementary modalities for cancer treatment, and may be combined to elicit synergistic effects. Recent years have witnessed the rapid development of small molecule-based cancer immunotherapy. In this review, we describe the current progress in small molecule-based immunomodulators (inhibitors/agonists/degraders) for cancer therapy, including those targeting PD-1/PD-L1, chemokine receptors, stimulator of interferon genes (STING), Toll-like receptor (TLR), etc. The tumorigenesis mechanism of various targets and their respective modulators that have entered clinical trials are also summarized.

Copper-Mediated Decarboxylative Coupling of 3-Indoleacetic Acids with Pyrazolones.

A copper-mediated decarboxylative coupling reaction of 3-indoleacetic acids with pyrazolones was described. This protocol realized new functionalization of pyrazolones under simple reaction conditions and exhibited high functional group compatibility and broad substrate scope. Notably, the products displayed antiproliferative activity against cancer cells.

Copper-Mediated Decarboxylative Coupling between Arylacetic Acids and 1,3-Dicarbonyl Compounds.

A copper-mediated decarboxylative coupling reaction between arylacetic acids and 1,3-dicarbonyl compounds was described. Significantly, methanocycloocta[b]indoles were also obtained by sequential intramolecular dehydrocyclization process in some cases. This protocol featured a broad substrate scope, simple operations, and good yields. Moreover, the products exhibited potent antiproliferative activity against the human cancer cell lines by a MTT assay.

Metal-free synthesis of 1,4-benzodiazepines and quinazolinones from hexafluoroisopropyl 2-aminobenzoates at room temperature.

Herein, we describe the novel reactivity of hexafluoroisopropyl 2-aminobenzoates. The metal-free synthesis of 1,4-benzodiazepines and quinazolinones from hexafluoroisopropyl 2-aminobenzoates has been developed at room temperature. These procedures feature good functional group tolerance, mild reaction conditions, and excellent yields. The newly formed products can readily be converted to other useful N-heterocycles. Moreover, the products and their derivatives showed potent anticancer activities in vitro by MTT assay.

Copper-Mediated Decarboxylative Sulfonylation of Arylacetic Acids with Sodium Sulfinates.

Herein, we present a copper-mediated decarboxylative sulfonylation of arylacetic acids with sodium sulfinates that provides viable access to sulfone compounds. This protocol features readily available feedstocks, simple operations, high regioselectivities, and moderate to good yields. The newly obtained products could be converted to other useful compounds. Importantly, the products and their derivatives exhibited potent antitumor activities in vitro, which were tested by MTT assay.