Treatment of castration-resistant prostate cancer (CRPC) remains challenging due to the development of drug resistance. The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose compared to normal tissues. We identified derivatives of natural 1,4-naphthoquinones to be active in CRPC and further synthetically modified them via glucose conjugation to increase selectivity by Warburg effect targeting. Mechanisms of action were examined by quantitative proteomics followed by bioinformatical analysis and target validation. Four synthesized molecules revealed the highest selectivity towards human CRPC cells, which correlated with higher GLUT-1 activity and expression. The compounds were able to induce pro-apoptotic signs and to inhibit the pro-survival processes and mechanisms of drug resistance (i.e., AR-signaling and autophagy). Proteome analysis suggested a disruption of the mitochondria/oxidative phosphorylation, which was validated by further functional analysis: thus, mitochondria depolarization, elevated levels of cytotoxic ROS, an increase of Bax/Bcl-2 ratio as well as release of mitochondrial AIF and cytochrome C to cytoplasm were observed. In conclusion, glucose-conjugated 1,4-naphthoquinones show potent activity and selectivity in human CRPC exerted via mitochondrial targeting. The compounds can overcome drug resistance against current standard therapies and suppress pro-survival mechanisms. This unique combination of properties makes them new promising candidates for the treatment of CRPC.

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The phenomenon of high sugar consumption by tumor cells is known as Warburg effect. It results from a high glycolysis rate, used by tumors as preferred metabolic pathway even in aerobic conditions. Targeting the Warburg effect to specifically deliver sugar conjugated cytotoxic compounds into tumor cells is a promising approach to create new selective drugs. We designed, synthesized, and analyzed a library of novel 6-S-(1,4-naphthoquinone-2-yl)-d-glucose chimera molecules (SABs)—novel sugar conjugates of 1,4-naphthoquinone analogs of the sea urchin pigments spinochromes, which have previously shown anticancer properties. A sulfur linker (thioether bond) was used to prevent potential hydrolysis by human glycoside-unspecific enzymes. The synthesized compounds exhibited a Warburg effect mediated selectivity to human prostate cancer cells (including highly drug-resistant cell lines). Mitochondria were identified as a primary cellular target of SABs. The mechanism of action included mitochondria membrane permeabilization, followed by ROS upregulation and release of cytotoxic mitochondrial proteins (AIF and cytochrome C) to the cytoplasm, which led to the consequent caspase-9 and -3 activation, PARP cleavage, and apoptosis-like cell death. These results enable us to further clinically develop these compounds for effective Warburg effect targeting.

Actinomyces naeslundii is a pioneer of the oral cavity and forms a biofilm on the tooth's surface. Most bacteria require iron for survival and in pathogenic bacteria iron availability regulates virulence gene expression. Metal-dependent repressors control gene expression involved in metal transport and uptake including siderophores. Siderophores are small molecules synthesized by bacteria and fungi to acquire iron. The A. naeslundii genome was searched for a gene encoding a metal-dependent repressor. Actinomyces metal-dependent repressor or amdR was identified. The AmdR protein was examined for its ability to bind to the promoter sequence of a gene encoding the siderophore uptake (sid gene). According to gel shift assays, AmdR binds to the sid gene promoter sequences. In the authors' model, when iron is available AmdR binds to the sid promoter and represses sid gene expression. To further explore the role of AmdR, an amdR-defective strain of A. naeslundii was constructed and biofilm formation and siderophore production were evaluated. When iron is removed from the medium A. naeslundii increases biofilm and siderophore production. However, amdR-defective A. naeslundii is less sensitive to metal ion concentrations in the growth medium.

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