Research paperSynthesis and structure-activity relationship studies of original cyclic diadenosine derivatives as nanomolar inhibitors of NAD kinase from pathogenic bacteria
Graphical abstract
Introduction
Antibacterial multidrug resistance is a major public health problem requiring urgent development of new antibiotics and identification of original bacterial targets, as pointed out by the World Health Organization [1]. We have initiated a decade ago the characterization of such a new target involved in NAD biosynthesis: the nicotinamide adenine dinucleotide kinase. NAD kinases (NADK, EC 2.7.1.23) catalyse the phosphorylation of NAD to NADP, which is subsequently reduced to NADPH [2,3]. As the sole known enzyme producing NADP de novo, NADK plays a crucial role in controlling the intracellular balance of NAD(H) and NADP(H) in many metabolic pathways [[4], [5], [6]]. NADK enzymatic properties have been known for decades [7], but the genes encoding the NADK were identified more recently, leading to the discovery of orthologs in nearly all living organisms [6,8,9]. The NADKs belong to a superfamily of kinases, including 6-phosphofructokinases (PFKs), soluble diacylglyceride kinases, and sphingosine kinases [10]. In line with a low overall sequence similarity (∼20% sequence identity), the human cytoplasmic and mitochondrial enzymes exhibit substantial differences compared to their bacterial counterparts [6,8,9]. Because of the essential role of NADK in all bacteria tested so far, such as Bacillus subtilis [11], Mycobacterium tuberculosis [12,13] and Staphylococcus aureus [14,15], NADK is considered as an attractive target for the development of novel antibiotics with an original mode of action [5,[16], [17], [18]].
During the last decade, we have developed a series of adenosine derivatives as potent inhibitors of recombinant Listeria monocytogenes and S. aureus NADKs [15,[19], [20], [21]]. The most potent molecules were found to bind to the NAD-binding site of NADK in a restricted conformation as compared to NAD thanks to the propargyl linker. One of these compounds became the first NADK inhibitor (NKI1, Fig. 1) active in mice infected with S. aureus, including antibiotic-resistant strains [15].
We next described the synthesis of a series of functionalized NKI1 derivatives as chemical tools to gain detailed knowledge about the mechanism of action of this lead compound [22]. Two of these new compounds harbor some chemical substitutions suggesting that macrocyclization could be envisioned to strengthen further their affinity and specificity for NADKs. The peculiar binding mode of NAD and its analogues into the active site of NADKs suggested that a more constrained compound would gain both affinity for those NADKs and specificity over a wide range of NAD(P) binding proteins [23,24]. Indeed, macrocyclization has been demonstrated to provide ligands with higher affinity due to entropy gain upon binding [[25], [26], [27]]. This was successfully implemented for several therapeutic targets [[28], [29], [30], [31]], but not yet applied to NADKs.
Recent progress have been made to develop cyclization strategies for macrocyclization of peptides [26], nucleosides [32] and oligonucleotides [33] or to generate libraries of macrocyclic compounds [34]. However, the synthesis of macrocycles is generally challenging since the ring-closing reaction efficiency depends on the size and geometry of the bridging linker. Thus, cyclization reactions do not favor the formation of large rings. Moreover, inter-instead of intra-molecular reaction is a major issue, in particular for the head-to-tail cyclization of peptides [30]. Therefore, most cyclizations need to be conducted in dilute solutions to favor macrocyclization over side product formation (dimerization or oligomerization). The ring-closing step can be achieved by a variety of chemical reactions, including amide coupling, ring-closing metathesis, nucleophilic substitution, palladium-catalyzed cross coupling reaction. The careful choice of ring disconnection and appropriate precursors can improve yields and reduce side reactions [27,29].
In the present study, we describe the synthesis of six original macrocycles (MC1-MC6, Fig. 2) inspired by linear derivatives of compound NKI1 [15,22]. These cyclic molecules differ in the functional group introduced at the 5′ position of one adenosine moiety (sulfonamide, amide, urea) and in the length of the small linking alkyl chain (5–8 atoms long).
The precise binding mode of the macrocycles MC1-6 into L. monocytogenes NADK (LmNADK1) was verified by X-ray crystallography. Their inhibitory potencies were measured in vitro on purified enzymes and evaluated on bacterial growth of two human pathogenic bacteria, L. monocytogenes and S. aureus. The structure-activity relationship study of this new class of inhibitors is presented hereafter.
Section snippets
Crystal structure of LmNADK1 in complex with a new linear diadenosine compound
As an amide moiety at 5′-end of NKI1 has been recently shown to fit quite well into the active site of LmNADK1 (PDB ID 6Z65) [22], we wondered if a sulfonamide group could accommodate as well. Such a NKI1 derivative (hereafter compound 1, Fig. 3A) was synthesized and successfully observed bound to LmNADK1 by X-ray crystallography. The structural analysis of LmNADK1 in complex with 1 highlighted key interactions and a special orientation of its amino-alkyl side chain in the NAD binding site of Lm
Conclusion
New compounds were designed from the various crystal structures of linear diadenosine derivatives bound to LmNADK1, synthesized, and tested in vitro against this still orphan drug target. A series of adenosine-based macrocyclic compounds were successfully obtained through ring closure reaction involving an amide bond formation. The acyclic precursors that contained the complementary functional groups (carboxylic acid and amine) were designed to minimize the building blocks required for
Structure-based design of novel cyclic diadenosines
Virtual molecules were designed by derivation from the compound NKI1. 3D conformers were generated from SMILE strings using Grade (http://grade.globalphasing.org/). The resulting compounds were docked in the electron density of NKI1 and minimized in various conformations compatible with the environment using COOT [40]. Virtual docking was also performed using PLANTS [41]. The results were compared to extract the most plausible conformations. Interactions with the neighboring residues were
Accession codes
The crystal structures of LmNADK1 bound to the macrocycles MC1-6 are deposited under accession codes PDB: 8B47, 7ZZH, 7ZZJ, 7ZZF, 7ZZG and 7ZZE, respectively. The complexes of LmNADK1 with the acyclic precursors are available in the PDB under the accession codes: 7ZZ7, 7ZZ9, 7ZZA, 7ZZB, 7ZZC, 7ZZD and 8A9V.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was funded by the Agence Nationale de la Recherche (NADKiller, ANR-17-CE18-0011-02) and supported by Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM) and University of Montpellier. C.L. and D.A.C. acknowledge ANR for financial support. This work was also supported by the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INSB-05-01). We acknowledge the experimental
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Present address: Institut Pasteur, Université Paris Cité, CNRS UMR3523, Unité de Chimie Biologique Epigénétique, F-75015 Paris, France.