Identification of NAE Inhibitors Exhibiting Potent Activity in Leukemia Cells: Exploring the Structural Determinants of NAE Specificity

Abstract

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

Inhibition of Neddylation Represses Lipopolysaccharide-induced Proinflammatory Cytokine Production in Macrophage Cells

“…Furthermore, the use of MLN4924, or other inhibitors of this molecular process, would represent a novel use for these pharmacological agents. MLN4924 is a sulfamated analog of AMP with an aminoindane group at position N6 of the deazapurine base (23), and a recent report has identified another neddylation inhibitor composed of a sulfamated AMP analog containing a N-hexyl group located at the same carbon position of the purine base (38). This C6 N-alkylated AMP analog inhibited neddylation at ϳ10 nM, which is a similar inhibitory concentration to that of MLN4924 in vitro (23,38).…”

“…MLN4924 is a sulfamated analog of AMP with an aminoindane group at position N6 of the deazapurine base (23), and a recent report has identified another neddylation inhibitor composed of a sulfamated AMP analog containing a N-hexyl group located at the same carbon position of the purine base (38). This C6 N-alkylated AMP analog inhibited neddylation at ϳ10 nM, which is a similar inhibitory concentration to that of MLN4924 in vitro (23,38). It would be interesting to determine whether this alkylated AMP analog also represses LPS-induced cytokine up-regulation in macrophage cells similarly to MLN4924.…”

TAS4464, A Highly Potent and Selective Inhibitor of NEDD8-Activating Enzyme, Suppresses Neddylation and Shows Antitumor Activity in Diverse Cancer Models

“…Although several studies have sought to identify NAE inhibitors as new potent anticancer agents, only MLN4924/TAK-924 (pevonedistat) has been formally clinically developed as an NAE inhibitor (15,23,24). On the basis of the preclinical evidence (8,(25)(26)(27), clinical trials of MLN4924 have been conducted to examine its efficacy in solid and hematologic tumors with various dosing regimens.…”

Natural Product Primary Sulfonamides and Primary Sulfamates

“…In this assay, compound 4 and its derivatives with small alkyl groups displayed good inhibitory activities (>16% inhibition at 1 nM). 42 Additional bioactivities that have been investigated for sulfamate 4 include in vitro inhibition of each of Mycobacterium tuberculosis FadD28 (fatty acid adenylating enzyme), 43 M. tuberculosis adenylate-forming enzyme MbtA, 44 siderophore biosynthesis in M. tuberculosis, 45 and histidine triad proteins (enzymes that act as hydrolases/transferases on nucleoside monophosphate substrates). In all cases, low activity was observed for 4, while analogues substituted on the sulfamate nitrogen of 4 showed good activity.…”

“…6,42,47 To synthesize sulfamates 4 and 5, the 2′,3′-hydroxy-groupprotected nucleosides 38 and 39 were prepared from the parent nucleosides, 36 and 37, respectively. 6 Next, sulfamoylation using sulfamoyl chloride together with a strong nonnucleophilic base (such as NaH) provided the sulfamoylated intermediates 40 and 41.…”