Elucidating the Unconventional Binding Mode of a DNA-Encoded Library Hit Provides a Blueprint for Sirtuin 6 Inhibitor Development.

Sirtuin 6 (Sirt6), an NAD+-dependent deacylase, has emerged as a promising target for aging-related diseases and cancer. Advancing the medicinal chemistry of Sirt6 modulators is crucial for the development of chemical probes aimed at unraveling the intricate biological functions of Sirt6 and unlocking its therapeutic potential. A proprietary DNA-encoded library yielded Sirt6 inhibitor 2-Pr, displaying remarkable inhibitory activity and isoform-selectivity, and featuring a chemical structure distinct from reported Sirt6 modulators. In this study, we explore the inhibitory mechanism of 2-Pr, evaluating the impact of chemical modifications and presenting a crystal structure of the Sirt6/ADP-ribose/2-Pr complex. Notably, co-crystal structure analysis reveals an unexpected and unprecedented binding mode of Sirt6, with 2-Pr spanning the acyl channel of the enzyme, extending into the acetyl-lysine binding pocket, and reaching toward the C-site. This unique binding mode guides potential avenues for developing potent and selective Sirt6 inhibitors.

DNA damage, cell cycle perturbation and cell death by naphthalene diimide derivative in gastric cancer cells.

Stomach cancer causes the third-highest cancer-related deaths worldwide. Limited availability of anticancer measures with higher efficiency and low unwanted toxicities necessitates the development of better cancer chemotherapeutics. Naphthalene diimide (NDI) derivatives have gained significant attention owing to their excellent anticancer potential. We evaluated the anticancer properties of NDI derivatives, 1a and 2a in cancer cell lines and found that 1a showed higher efficacy as compared to 2a exhibiting a remarkable difference in activity upon single atom substitution of C with N. Particularly, NDI 1a showed potent inhibitory activity against gastric cancer cell line AGS with IC50 of 2.0 µM. NDI 1a induced remarkable morphological changes and reduced clonogenicity as well as the migratory ability of AGS cells. The reduction in AGS cell migration was mediated through inhibition of Tyr397 p-FAK dephosphorylation at focal adhesion points leading to enhanced attachment of cells at contact points. NDI 1a caused extensive DNA double-strand-breaks (DSBs) leading to activation of p53 and its transcriptional target p21. Reduced nuclear BRCA1 but enhanced nuclear p53BP1 foci formation upon 1a treatment suggests that DNA DSB repair is mediated through error-prone NHEJ which led to the accumulation of extensive DNA damage. Combinatorial effects mediated by interactions of 1a with double-stranded DNA through minor groove binding as well as induction of intracellular ROS exacerbated the loss of genomic integrity induced by 1a. NDI 1a mediated DNA damage-induced S phase arrest; however, cells experiencing extensive and irreparable DNA damage underwent mitochondrial apoptosis through downregulation of anti-apoptotic protein p21. Furthermore, proliferation inhibitory activity of 1a is also attributed to inhibition of ß-catenin/c-Myc axis in AGS cells with constitutively active ß-catenin pathway. In vivo toxicity analysis of 1a revealed minimal systemic toxicity suggesting that compound 1a is a safe and potential candidate for the development of gastric cancer chemotherapeutics.

Synthesis of Novel Ciprofloxacin-Based Hybrid Molecules toward Potent Antimalarial Activity.

Antimalarial drug resistance is a serious obstacle in the persistent quest to eradicate malaria. There is a need for potent chemical agents that are able to act on drug-resistant Plasmodium falciparum populations at reasonable concentrations without any related toxicity to the host. By rational drug design, we envisaged to address this issue by generating a novel hybrid drug possessing two pharmacophores that can act on two unique and independent targets within the cell. We synthesized a new class of ciprofloxacin-based hybrid molecules, which have been integrated with acridine, quinolone, sulphonamide, and cinnamoyl pharmacophores (1-4). We realized a potent chloroquinolone-ciprofloxacin-based antimalarial hybrid (2, CQ-CFX) whose mechanism of action is unlike that of its parent molecules indicating a unique biological target. CQ-CFX is not only potent against CQ-resistant and susceptible strains of Plasmodium falciparum at low nanomolar concentrations (IC50 values are 63.17 ± 1.2 nM and 25.52 ± 4.45 nM, respectively) but is also not toxic to mammalian and bacterial systems up to 20 µM and 1 µM, respectively.

A Focused DNA-Encoded Chemical Library for the Discovery of Inhibitors of NAD+-Dependent Enzymes.

DNA-encoded chemical libraries are increasingly used in pharmaceutical research because they enable the rapid discovery of synthetic protein ligands. Here we explored whether target-class focused DNA-encoded chemical libraries can be cost-effective tools to achieve robust screening productivity for a series of proteins. The study revealed that a DNA-encoded library designed for NAD+-binding pockets (NADEL) effectively sampled the chemical binder space of enzymes with ADP-ribosyltransferase activity. The extracted information directed the synthesis of inhibitors for several enzymes including PARP15 and SIRT6. The high dissimilarity of NADEL screening fingerprints for different proteins translated into inhibitors that showed selectivity for their target. The discovery of patterns of enriched structures for six out of eight tested proteins is remarkable for a library of 58 302 DNA-tagged structures and illustrates the prospect of focused DNA-encoded libraries as economic alternatives to large library platforms.

Downregulation of c-Myc and p21 expression and induction of S phase arrest by naphthalene diimide derivative in gastric adenocarcinoma cells.

Naphthalene diimide (NDI) derivatives have been shown to exhibit promising antineoplastic properties. In the current study, we assessed the anticancer and anti-bacterial properties of di-substituted NDI derivative. The naphthalene-bis-hydrazimide, 1, negatively affected the cell viability of three cancer cell lines (AGS, HeLa and PC3) and induced S phase cell cycle arrest along with SubG0/G1 accumulation. Amongst three cell lines, gastric cancer cell line, AGS, showed the highest sensitivity towards the NDI derivative 1. Compound 1 induced extensive DNA double strand breaks causing p53 activation leading to transcription of p53 target gene p21 in AGS cells. Reduction in protein levels of p21 and BRCA1 suggested that 1 treated AGS cells underwent cell death due to accumulation of DNA damage as a result of impaired DNA damage repair. ß-catenin downregulation and consequently decrease in levels of c-Myc may have led to 1 induced AGS cell proliferation inhibition.1 induced AGS cell S phase arrest was mediated through CylinA/CDK2 downregulation. The possible mechanisms involved in anticancer activity of 1 includes ROS upregulation, induction of DNA damage, disruption of mitochondrial membrane potential causing ATP depletion, inhibition of cell proliferation and downregulation of antiapoptotic factors ultimately leading to mitochondria mediated apoptosis. Further compound 1 also inhibited H. pylori proliferation as well as H. pylori induced morphological changes in AGS cells. These findings suggest that NDI derivative 1 exhibits two-pronged anticancer activity, one by directly inhibiting cancer cell growth and inducing apoptosis and the other by inhibiting H. pylori.

Design, Synthesis and Evaluation of Bifunctional Acridinine-Naphthalenediimide Redox-Active Conjugates as Antimalarials.

A novel class of bifunctional molecules was synthesized integrating acridine (Ac) and redox-active naphthalenediimide (NDI) scaffolds directly and through a flexible linker (en). We evaluated in vitro antiplasmodial activity, physicochemical properties, and a possible mode of action. Theoretical studies suggested electronic segmentation between the electron-rich Ac and electron-deficient NDI scaffolds. Orthogonal Ac-NDI molecules showed activities in the micromolar to submicromolar range against a chloroquine (CQ)-sensitive strain of human malaria pathogen Plasmodium falciparum (maximum activity, IC50: 0.419 µM). The flexible Ac-en-NDI molecules were most potent and showed activity in the nanomolar range against both CQ-sensitive (with most effective compounds, IC50: 3.65 and 4.33 nM) as well as CQ-resistant (with most effective compounds, IC50: 52.20 and 28.53 nM) strains of P. falciparum. Significantly, with CQ-resistant strains, the activity of the most effective compounds was 1 order of magnitude better than that of standard drug CQ. Ac-en-NDI-conjugated molecules were significantly more potent than the individual NDI and Ac-based molecules. The structure-activity relationship (SAR) suggests that the flexible spacer (en) linking the Ac and NDI scaffolds plays a vital role in exhibiting improved potency. None of the molecules triggered hemolysis in culture, and the most potent compounds did not show cytotoxicity in vitro against mammalian fibroblast NIH3T3 cells at their respective IC50 values. The other significant outcome of this work is that some of the investigated molecules have the potential to affect multiple processes in the parasite including the hemozoin formation in digestive vacuoles (DVs), mitochondrial membrane potential, and the redox homeostasis of the parasite.

Assorted morphosynthesis: access to multi-faceted nano-architectures from a super-responsive dual π-functional amphiphilic construct.

An electronically segmented amphiphile was created by conjugating two π-functional units hydroxyquinoline and naphthalenediimide (HQ/NDI) for the first time. The differential electrostatic potential of the π-surfaces, H-bonding units, etc. trigger a manifold response and direct the assembly of a unique collection of seven diverse nano-architectures. Chiral assembly, distinct classes of fibers, 3-D sheets, and metallo-spheres/fibrils with µM levels of Co/Cu/Zn(ii) ions emerged from this new approach of assorted morphosynthesis under ambient conditions.

Potent antimalarial activity of acriflavine in vitro and in vivo.

Malaria continues to be a major health problem globally. There is an urgent need to find new antimalarials. Acriflavine (ACF) is known as an antibacterial agent and more recently as an anticancer agent. Here, we report that ACF inhibits the growth of asexual stages of both chloroquine (CQ) sensitive and resistant strains of human malarial parasite, Plasmodium falciparum in vitro at nanomolar concentration. ACF clears the malaria infection in vivo from the bloodstreams of mice infected with Plasmodium berghei. Interestingly, ACF is accumulated only in the parasitized red blood cells (RBCs) and parasite specific transporters may have role in this specific drug accumulation. We further show that ACF impairs DNA replication foci formation in the parasites and affects the enzymatic activities of apicoplast specific Gyrase protein. We thus establish ACF as a potential antimalarial amidst the widespread incidences of drug resistant Plasmodium strains.

Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast.

Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single-stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena.