Click Chemistry for the development of enzyme inhibitors (II)

1. Glycosidase inhibitor

Glycoside hydrolases is an enzyme that catalyzes the hydrolysis of glycosidic bonds in oligosaccharides and sugar derivatives. Its main function is to participate in metabolism and cell-cell, cell-virus recognition processes. Finding effective inhibitors of glycosidase is the key to the treatment of many diseases such as diabetes, viral infections, lysosomal storage disorders and tumor metastasis. Under physiological conditions, imino sugars have the ability to mimic the transition state of corresponding oxygen carbocations or O-glycoside hydrolysis catalyzed by enzymes, and can occupy corresponding enzyme active sites. They are the most important type of glycosidase inhibitors. Studies have shown that the introduction of aromatic heterocycles on monocyclic imino sugars can increase the interaction of non-sugar moieties with enzymes, increase the lipophilicity required for drugs to pass through cell membranes, and obtain glycosidase inhibitors with stronger potency and selectivity. Based on these understandings, a library of pyrrolizine-aromatic derivatives was established through different click reactions (formation of urea, thiourea and triazole), and combined with in-situ biological screening, highly active glycosidase inhibitor compounds 11-13 were quickly and effectively screened out. The triazole derivative 11 exhibits moderate inhibitory effect on α-L fucosidase and α-galactosidase, and has a higher inhibitory effect on α-mannosidase, which is a multi-targeted glycosidase inhibitor; while urea analogs 12 and thiourea derivatives 13 are more selective α-galactosidase and α-mannosidase inhibitors. Molecular docking studies have shown that the triazole part of compound 11 can interact with the glycosidase binding site by hydrogen bonding, thereby enhancing its inhibitory effect. This study identified a new type of α-glucosidase inhibitor through in situ click chemistry, which enriched the choice of lead compounds for α-glucosidase inhibitors.

1. O-GlcNAc transferas inhibitor

O-GlcNAc transferase (OGT) mainly regulates the glycosylation process in the organism, and realizes the glycosylation processing by transferring the active glycosyl part of the corresponding glycosyl donor to sugars, proteins, lipids and nucleic acids, etc. Tumor cell proliferation and invasion, angiogenesis and immune regulation are all related to abnormal glycosylation process. Therefore, the expression and activity of glycosyltransferases including N-acetylglucosaminyltransferase and fucosyltransferase can be used as diagnostic markers of tumorigenesis. At the same time, the increased activity of some glycosyltransferases is also considered to be an important cause of tumor migration and deterioration. The development of highly effective glycosyltransferase inhibitors is of great significance for the search for new anti-tumor and anti-immune drugs.

How to synthesize a series of new OGT inhibitors through tethering in situ click chemistry (TISCC)? Firstly, the azide building block and the target protein are incubated together to achieve the “bundling” of the target protein and the building block, and then add an alkynyl building block to generate an in-situ click reaction, and separate the protein and small molecules by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and find the modified peptide through the screening of modified peptides, and finally, the two OGT inhibitor compounds 14 and 15 that are permeable to cells were found. Both of these compounds can reduce the level of O-GlcNAc glycosylation in cells without obvious cytotoxicity. Compared with classic in situ click chemistry, TISCC uses more general building blocks, and only needs to detect protein fragments, that is, multiple binding pockets can be screened at the same time. The discovery of compounds 14 and 15 shows that TISCC is a kind of new lead compound and a more efficient in-situ click chemistry method for components with weaker molecular binding.

1. HIV-1 non-nucleoside reverse transcriptase inhibitor

HIV-1 reverse transcriptase (HIV-1 RT) is one of the important targets for the development of anti-HIV drugs. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are used in highly active antiretroviral therapy (HAART) for the treatment of HIV due to their unique antiviral activity, high specificity and low toxicity. NNRTIs interact with HIV-1RT's non-nucleoside inhibitory binding pocket (NNIBP) in a non-competitive manner to induce conformational changes in the catalytic domain, thereby inhibiting its DNA polymerase activity and exerting anti-HIV effect.

Early studies proposed a new type of potent thiophene [3,2-d] pyrimidine nucleoside 16, and the co-crystal structure of HIV-1 wild-type (wild-type, WT) reverse transcriptase complex showed that compound 16has significant structural complementarity with NNIBP when used in NNIBP horseshoe conformation, and has a large amount of extension in the three channels of hydrophobic channel, tolerance zone I and tolerance zone II. The thiophene [3,2-d] pyrimidine heterocyclic 16 structure is located in the tolerance zone II formed by Glu138 and Lys101, and forms a new non-polar interaction with the alkyl chain of Glu138. In addition, the nitrogen atom in the central thiophene pyrimidine and the amine group connecting the central thiophene pyrimidine and piperidine ring form a double hydrogen bond with the carbonyl oxygen of Lys101, which plays an important role in maintaining its high anti-HIV activity.

In subsequent studies, the researchers used thiophene [3,2-d] pyrimidine heterocyclic 16 as the lead compound to derive different alkyne building blocks, and then designed different azide substituents according to the spatial structure of NNIBP, and obtained compounds 17 and 18 through in-situ click reaction screening. The results of molecular docking studies show that compounds 16, 17, and 18 bind to WT-HIV-1 in a similar manner. The sulfa group of the lead compound 16 can form a hydrogen bond with Val-106, while the amide group of 17 only has a hydrogen bond interaction with Lys-104, and the morpholine ring in 18 does not produce any hydrogen bond interaction. This may be due to their decreased activity of WT-HIV-1. For the more destructive RES056 double mutant HIV reverse transcriptase, the mutation eliminates the most favorable π-π interaction between 17 and Tyr-181. In addition, the terminal substituent is twisted and loses its hydrogen bond with Lys-104. Although the triazole group forms a new hydrogen bond with the mutant Asn-103 through water molecules, it is not enough to compensate for the loss of π-π interaction and the hydrogen bond with Lys-104. All of these may be the reason for the decreased anti-HIV-1 activity. By comparing the binding modes of 17 and 18 with WT and RES056 reverse transcriptase, it was found that triazole plays a key role in NNIBP, proving that triazole is the dominant segment of this type of inhibitor. The in-situ click reaction provides a new guiding idea for further research and development of the hydrophobic channel and tolerance zone of NNIBP, and the discovery of new NNRTIs with greater potential.

1. Chitinase inhibitor

Chitinase is a chitinase hydrolase whose main function is to catalyze the hydrolysis of chitin to N-acetylglucosamine. Chitin, also known as chitin, is the second most abundant polysaccharide in nature. It is composed of glucosamine units linked by N-amino-acetyl-D-aspartic acid and is the main component of various biological cell walls and exoskeletons. Since the synthesis and metabolism of chitin are important factors for maintaining the life and reproduction of invertebrates, chitinase inhibitors have potential applications for pesticides and antifungal drugs. Using chitinase as the target enzyme, the corresponding derivative 19 is obtained by modifying arginine with azide and the alkyne 20 is subjected to in-situ click reaction to obtain compound 21, which exhibits resistance to Serratia marcescens chitinase B (SmChiB). Compared with the natural product cyclopenta peptide inhibitor arginine, the inhibitory effect of compound 21 on SmChiB was increased by about 300 times. Chemical calculations show that SmChiB mainly promotes the in-situ click reaction through the cage effect, that is, the propelling effect of the protein increases the collision frequency between azide and alkynes, which is beneficial to the progress of the cycloaddition reaction.

In-situ click chemistry actively selects the best ligands through biological targets, effectively shortening the drug discovery process and providing strong support for drug development. In the research of enzyme inhibitors, in-situ click chemistry has been developed as an effective method for fragment-based inhibitor synthesis.

References

[1] THIRUMURUGAN P, MATOSIUK D, JOZWIAKK. Click chemistry for drug development and diverse chemical-biology applications[J]. Chem Rev, 2013, 113(7): 4905-4979.

[2] HIMO F, LOVELL T, HILGRAF R, et al. Copper(Ⅰ) -catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates[J]. Am Chem Soc, 2005, 127(1): 210-216.

[3] PRASHER P, SHARMA M. Tailored therapeutics based on 1, 2, 3-1H-triazoles: a mini review [J]. Med Chem Comm, 2019, 10(8): 1302-1328.

[4] BHARDWAJ A, KAUR J, WUEST M, et al. In situ click chemistry generation of cyclooxygenase-2 inhibitors[J]. Nat Commun, 2017, 8(1): 1-14

[5] MONDAL M, RADEVA N, FANLO-VIRGOS H, et al. Fragment linking and optimization of inhibitors of the aspartic protease endothiapepsin: fragment-based drug design facilitated by dynamic combinatorial chemistry[J]. Angew Chem Int Ed Engl, 2016, 55 (32): 9422-9426.

[6] WANG Y, ZHU J, ZHANG L. Discovery of cell-permeable O-GlcNAc transferase inhibitors via tethering in situ click chemistry[J]. J Med Chem, 2017, 60(1): 263-272.

[7] KANG D, FENG D, JING L, et al. In situ click chemistry-based rapid discovery of novel HIV-1 NNRTIs by exploiting the hydrophobic channel and tolerant regions of NNIBP[J]. Eur J Med Chem, 2020, 193: 112237.