Structure–Activity Relationship of 3-Methylcytidine-5′-α,β-methylenediphosphates as CD73 Inhibitors

Abstract

We recently reported N 4-substituted 3-methylcytidine-5′-α,β-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure–activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; K i = 0.673 nM) and 4-iodo (MRS4620 18; K i = 0.436 nM) substitution of the N 4-benzyloxy group decreased K i by ∼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N 4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N 4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Discovery of Novel Non-Nucleoside Inhibitors Interacting with Dizinc Ions of CD73

“…1 H NMR (400 MHz, chloroformd) δ 4.31 (q, J = 7.1 Hz, 4H), 1.32 (t, J = 7.1 Hz, 6H). (20). Compound 18 (1.20 g, 4.33 mmol) was dissolved with 200 mL of toluene, then 19 (1.20 g, 6.49 mmol) and rhodium acetate dimer (0.096 g, 0.22 mmol) were added.…”

“…19 Since the dephosphorylation products of the above nucleoside inhibitors are derivatives of adenosine, the strategy of MRS4620 ( 6) is to replace adenine with methylcytosine, thereby reducing the possibility of activating the adenosine pathway. 20 Therefore, non-nucleoside inhibitors have more significance in pharmacology. Among them, benzotriazole analogue 7b cocrystallized with CD73 in the closed conformation exhibits a competitive binding mode.…”

Molecular Dynamics Simulations of the Human Ecto-5′-Nucleotidase (h-ecto-5′-NT, CD73): Insights into Protein Flexibility and Binding Site Dynamics

“…Adenosine generated by overexpressed h-ecto-5′-NT accumulates in the tumor microenvironment, where it activates immunosuppressive pathways that favor the development of neoplasia. − Accumulated adenosine can also regulate angiogenesis and proliferation, migration, differentiation, and apoptosis of parenchymal cancer cells, contributing to tumor progression and metastasis. − The effects of h-ecto-5′-NT overexpression in tumorigenesis and its role in tumor immune escape and tumor metastasis have been shown in vivo . − In addition to its participation in tumor progression, h-ecto-5′-NT plays a central role in the course of several pathophysiological events and diseases, including inflammation and autoimmune, infectious, and neurological diseases. , Therefore, h-ecto-5′-NT has been recognized as a well-established biological target for cancer therapy and for the treatment of many other diseases. Several h-ecto-5′-NT inhibitors have been reported so far, ,− some of which are currently being tested in clinical trials. , Nevertheless, as discussed in the literature, ,, many of the known inhibitors have physicochemical and/or pharmacological characteristics that limit their applicability as drug candidates, such as low inhibitory potency, poor water solubility, and lack of selectivity. For this reason, considerable efforts have been made by the scientific community and the pharmaceutical industry to search for novel h-ecto-5′-NT inhibitors …”

“…The elucidation of the h-ecto-5′-NT 3D structure by X-ray crystallography (resolution values ranging from 1.05 to 2.90 Å), ,,,,,,,,− in its open and closed conformations (Figure ), has provided the basis for structure-based design of inhibitors of this target enzyme . The available crystal structures reveal that h-ecto-5′-NT consists of a homodimer, , in contrast to the monomeric bacterial 5′-nucleotidases. , As shown in Figure , each dimer subunit is formed by two structural domains: the C-terminal domain, which contains the substrate binding site, and the N-terminal domain, which contains the binding sites of two zinc ions that are essential for catalysis. , The dimerization interface is formed by C-terminal domain residues of each dimer subunit, which interact via a network of hydrogen-bonds and hydrophobic interactions .…”

Combination of multiple methods and views for recognition, transportation, and structure-guided modification of lysine-specific demethylase phenylcyclopropylamine inhibitor

“…34,35 In addition, molecular dynamics simulation has been widely employed to study the binding mode of potential inhibitors in the active site of the target for designing new compounds. 36,37 These studies provide valuable clues for drug design, while the MM MD simulations are usually used to capture static binding for the target ligand in the binding site. The inhibitor delivery process always involves its recognition and delivery, which may also influence its efficacy.…”