The rise of antibiotic resistant bacteria is a global threat for healthcare, making it possible to succumb to diseases that were previously treatable (Wong et al., 2017). This has been acknowledged by both the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), which generated a list of drug-resistant pathogens for which new antibiotics are urgently needed (World Health Organization, 2017; CDC, 2019). The top priorities of these lists are antibiotic-resistant Acinetobacter baumannii bacteria (Whiteway et al., 2021).

A. baumannii is a Gram-negative, opportunistic bacterium, belonging to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) of the most problematic nosocomial pathogens (Rice, 2008; Santajit and Indrawattana, 2016; Whiteway et al., 2021). While A. baumannii is a ubiquist (i.e. it can be found in soil, on human skin and in water sources), its presence especially imposes a threat in clinical settings (Vallenet et al., 2008; Wieland et al., 2018). This is due to a remarkable combination of resistance capabilities of A. baumannii, which is able to persist prolonged periods of desiccation, resist to disinfectants and to acquire drug resistance at a high rate (Da Silva and Domingues, 2016; Zeidler and Müller, 2019). Infections caused by A. baumannii commonly occur in immunocompromised patients and manifest as ventilator-assisted pneumonia, bacteremia and to a lesser extent skin or urinary tract infections (Weiner et al., 2016; Whiteway et al., 2021). Treatment of these infections becomes increasingly difficult, as multidrug-resistant, extensively drug-resistant or even pandrug-resistant strains have been reported, with the latter being resistant to all available antibiotics, including carbapenems (Magiorakos et al., 2012; Gallagher et al., 2015). An important hurdle in the development of new antimicrobials against A. baumannii is the high diversity found between isolates, leading to a still open pan-genome (Adams et al., 2008).

In this context, the discovery of new inhibitory molecules, active against multidrug-resistant A. baumannii strains, is therefore crucial (World Health Organization, 2017; CDC, 2019). Harmine and harmine derivatives are β-carbolines that present pharmacological effects including anti-inflammatory, neuroprotective, antidiabetic, and antitumor activities (Zhang et al., 2020). β-carbolines also show anti-infective potential (Faheem et al., 2021). For natural and synthetic β-carboline analogs, antibacterial, antifungal, antiviral, antimalarial, antileishmanial and antitrypanosomal properties have been reported. Most studies deal with biological evaluation of the molecules and, only in some rare cases, potential mechanisms of action have been proposed. Mechanistic studies demonstrated that harmine effectively repressed the HSV-2 induced upregulation of Interleukin-1β (IL-1β), Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) (Chen et al., 2015). Mechanistically, substituted β-carbolines exhibit their anti-infective properties via several targets, including Topoisomerase II, DNA gyrase, MAPK, PfHsp90 or trypanothione reductase (Faheem et al., 2021).

For example, it has been shown that harmine exerts antileishmanial activity both in vitro and in vivo and causes necrosis by a nonspecific membrane damage in Leishmania donovani promastigotes (Lala et al., 2004). Harmine inhibits P. falciparum heat shock protein 90 by specific competition with its ATP-binding domain (Shahinas et al., 2012). Harmine analogs were further studied as binders to P. falciparum heat shock protein 90 (PfHsp90) and two compounds inhibited the parasite in vitro at micromolar concentrations, reducing parasitemia, and prolonging the survival of P. berghei-infected mice (Bayih et al., 2016). Adequate substitution of the β-carboline scaffold led to enhanced nematicidal effect (Xia et al., 2019). Manzamine alkaloids, which are complex natural compounds consisting of a β-carboline nucleus, have been isolated and found to exhibit potent anti-infective activities by inhibiting target kinases like GSK-3β and MtSK (Ashoka et al., 2021).

9-substituted harmine derivatives show an inhibitory effect on dengue virus and impair the maturation and release of virus particles to the extracellular medium affecting the spreading of the infection (Quintana et al., 2016). Their mode of action still needs to be clarified. 9N-methylharmine neither affects viral adsorption-internalization events nor viral RNA synthesis. The antiviral activity is not related to the ability of the compound to downregulate p38 MAPK phosphorylation.

Harmine derivatives with 7,9- or 2,7,9-substituted 7-oxy-1-methyl-β-carboline scaffolds inhibit activity of SerB2, an essential metabolic enzyme and suspected virulence factor of Mycobacterium tuberculosis (Frédérick et al., 2012; Carvalho et al., 2017; Carvalho et al., 2020; Pierson et al., 2020).

In this paper, we aimed at the discovery of a compound active against most clinical isolates. We performed a repurposing screen on a compound library, which led to the identification of a harmine-derived compound, called HDC1, with inhibitory activity on the growth of all the tested clinical isolates, amongst which 40 are carbapenem-resistant.

In this study, a repurposing drug screen led to the discovery of a compound with inhibitory activity on the growth of a multidrug-resistant Acinetobacter baumannii reference strain, AB5075-VUB. This compound, named HDC1, is a harmine-derivative previously designed to have anticancer properties. The anticancer screen showed that the compound acts as a protein synthesis inhibitor at a concentration of 0.7 µM (Frédérick et al., 2012). In our study, HDC1 was shown to have bactericidal activity on AB5075-VUB with an IC₅₀ of 48.23 µM. As the compound was previously found to be cytotoxic in HEPG2 cells at 50 µM, this limits the potential of HDC1 as a new antimicrobial without further modification of the molecule (Marx et al., 2019). Differences in active concentrations of HDC1 between cancer and bacterial cells could be attributed to cell size and target(s) presence and/or abundance, altered metabolism states and the presence or absence of post-translational modifications. However, as multidrug-, extensively drug- and pandrug-resistant A. baumannii strains are emerging and spreading, every option deems to be explored.

Due to the high diversity between A. baumannii isolates, one of the main hurdles in the discovery of new compounds is to find compounds capable of targeting the majority of the isolates. Here, we report a compound to have inhibitory activity on all the tested and recent carbapenem-resistant A. baumannii isolates. Our test shows various degrees of growth inhibition: from complete to intermediate to only slight inhibition. In addition, the 4 reference strains used in our study all show a high degree of sensitivity to HDC1. Taken together, this raises the following questions (i) what contributes to this difference in growth inhibition levels, (ii) what could be the target(s) of HDC1 and (iii) why is the whole AB5075-VUB bacterial population not killed by HDC1 in the tested conditions, since a significant bactericidal effect is observed? The phase variation observed in AB5075 might be the answer to the last question. Phase variation is a morphological feature of A. baumannii colonies where 2 different opacity phenotypes are observed: opaque and translucent (Tipton et al., 2015). Opaque and translucent colonies exhibit multiple phenotypic differences, including cell morphology, surface motility, biofilm formation and virulence, but of particular interest also a difference in resistance to antibiotics and thus potentially to HDC1 (Tipton et al., 2015; Chin et al., 2018). Interestingly, a recent study showed that HDC1 inhibits the phosphoserine phosphatase of Mycobacterium tuberculosis, MtSerB2 (Pierson et al., 2020). MtSerB2 catalyzes the last step in the L-serine biosynthetic pathway and is involved in immune evasion mechanisms of M. tuberculosis (Pierson et al., 2020). In AB5075-VUB, a homolog of MtSerB2 is present: AbSerB. This indicates a putative target of HDC1 in A. baumannii. A possible explanation for the different growth inhibition profiles of the clinical isolates could be the presence of mutations in the serB gene. Although serB is highly conserved in all our tested A. baumannii strains (sequence identity between 98.45-100%), no correlation could be found between mutations in serB and the growth inhibition profiles of the A. baumannii isolates ( Supplementary Table 2 ). The target(s) of HDC1 in A. baumannii remain(s) to be determined. Interestingly, it has been shown for M. tuberculosis that certain compounds are more efficient at inhibiting growth of the bacterium itself, than inhibiting the enzyme only, suggesting different mechanisms of action or intracellular accumulation of the compounds (Haufroid and Wouters, 2019). Additional resistance mechanisms, potentially countering such effects, could explain the higher resistance to HDC1 of some clinical isolates. Nevertheless, HDC1 has broad activity on all the tested recent clinical A. baumannii isolates of our study, which prompts the further exploration of this compound and/or cognate putative target in A. baumannii for drug discovery.

In conclusion, HDC1 is a potent harmine-derived compound with antibacterial activity identified using a multidrug-resistant A. baumannii strain, that also significantly inhibits the growth of diverse, recent, and carbapenem-resistant clinical A. baumannii isolates.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Drafting of the manuscript, AB and CV. Corrections of the manuscript, AB, MV, ME, CP, JW, SB, and CV. HDC1 production, ME and SB. Preliminary drug screen, TQ, CP, and CV. Bioinformatics, AB and CH. Experiments, AB, CW, and CV. Data analyses, AB, MV, JW, and CV. All authors contributed to the article and approved the submitted version.

AB is recipient of a PhD fellowship Strategic Basic Research of the Research Foundation – Flanders (FWO, File number: 77258). ME and SB acknowledge financial support of the Research Council at the Vrije Universiteit Brussel (VUB) through the IRP funding scheme. CV acknowledges the financial support from the Flanders Institute for Biotechnology (VIB). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 748032.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.