Development and characterization of a new inhibitor of heme oxygenase activity for cancer treatment.

Heme oxygenase-1 (HO-1, HMOX1) degrades pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe2+) and biliverdin. The enzyme exerts multiple cytoprotective functions associated with the promotion of angiogenesis and counteraction of the detrimental effects of cellular stress which are crucial for the survival of both normal and tumor cells. Accordingly, in many tumor types, high expression of HO-1 correlates with poor prognosis and resistance to treatment, i.e. chemotherapy, suggesting inhibition of HO-1 as a possible antitumor approach. At the same time, the lack of selective and well-profiled inhibitors of HO-1 determines the unmet need for new modulators of this enzyme, with the potential to be used in either adjuvant therapy or as the stand-alone targeted therapeutics. In the current study, we provided novel inhibitors of HO-1 and validated the effect of pharmacological inhibition of HO activity by the imidazole-based inhibitor (SLV-11199) in human pancreatic (PANC-1) and prostate (DU-145) cancer cell lines. We demonstrated potent inhibition of HO activity in vitro and showed associated anticancer effectiveness of SLV-11199. Treatment with the tested compound led to decreased cancer cell viability and clonogenic potential. It has also sensitized the cancer cells to chemotherapy. In PANC-1 cells, diminished HO activity resulted in down-regulation of pro-angiogenic factors like IL-8. Mechanistic investigations revealed that the treatment with SLV-11199 decreased cell migration and inhibited MMP-1 and MMP-9 expression. Moreover, it affected mesenchymal phenotype by regulating key modulators of the epithelial to mesenchymal transition (EMT) signalling axis. Finally, F-actin cytoskeleton and focal contacts were destabilized by the reported compound. Overall, the current study suggests a possible relevance of the tested novel inhibitor of HO activity as a potential anticancer compound. To support such utility, further investigation is still needed, especially in in vivo conditions.

Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties.

In oncology, the "Warburg effect" describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.

Discovery and Structure-Activity Relationships of N-Aryl 6-Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors.

Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure-activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC50 of 14 nm for the target and an IC50 of 0.49 µm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.

SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains.

Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids as inhibitors of ureases.

Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (Ki=13±0.8 and 0.62±0.09 µM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique.

Exploring the Sn binding pockets in gingipains by newly developed inhibitors: structure-based design, chemistry, and activity.

Arg-gingipains (Rgps) and Lys-gingipain (Kgp) are cysteine proteinases secreted by Porphyromonas gingivalis, the major pathogen implicated in periodontal disease. Gingipains are essential to the bacterium for its virulence and survival, and development of inhibitors targeting these proteins provides an approach to treat periodontal diseases. Here, we present the first example of structure-based design of gingipains inhibitors, with the use of the crystal structure of RgpB and the homology model of Kgp. Chloromethyl ketones were selected as suitable compounds to explore the specificity of the Sn binding region of both enzymes. Three series of inhibitors bearing Arg or Lys at P1 and different substituents at P2 and P3 were designed, synthesized, and tested. High potency (k(obs)/[I] approximately 10(7) M(-1) s(-1)) was achieved for small ligands, such as the dipeptide analogues. The detailed analysis of Sn binding pockets revealed the molecular basis of inhibitory affinity and provided insight into the structure-activity relationship.