Polycyclic polyprenylated acylphloroglucinols (PPAPs) comprise a large group of compounds of mostly plant origin. The best-known compound is hyperforin from St. John's wort with its antidepressant, antitumor and antimicrobial properties. Chemical synthesis of PPAP variants allows the generation of compounds with improved activity and compatibility. Here we studied two synthetic PPAP-derivatives, PPAP 23 and PPAP 53, as to their antimicrobial activity. While PPAP 23 is insoluble in water, PPAP 53, the sodium salt, is water-soluble. In vitro, both compounds exhibited good and comparable activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Both compounds had no adverse effects on Galleria mellonella wax moth larvae, even at high concentrations. However, they were unable to protect the larvae from infection with S. aureus because components of the larval coelom neutralized the antimicrobial activity; a similar neutralizing effect was also seen with serum albumin. In a septic arthritis model triggered by S. aureus PPAP23 decreased the formation of abscesses and bacterial load in kidneys, and in a mouse skin abscess model topical treatment with PPAP 53 resulted in an approximately two-fold reduction in S. aureus counts. PPAP 23 and PPAP 53 also showed high growth inhibiting activity against anaerobic Gram-positive pathogenic gut bacteria such as Clostridium perfringens and Clostridium difficile. Based on these results, we envision potential application of PPAP 23 and PPAP 53 in the topical treatment of wound infections with Gram+ pathogens, and their oral use in intestinal infections with C. difficile.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) comprise a large group of compounds of mostly plant origin. The best-known compound is hyperforin from St. John’s wort with its antidepressant, antitumor and antimicrobial properties. The chemical synthesis of PPAP variants allows the generation of compounds with improved activity and compatibility. Here, we studied the antimicrobial activity of two synthetic PPAP-derivatives, the water-insoluble PPAP23 and the water-soluble sodium salt PPAP53. In vitro, both compounds exhibited good activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Both compounds had no adverse effects on Galleria mellonella wax moth larvae. However, they were unable to protect the larvae from infection with S. aureus because components of the larval coelom neutralized the antimicrobial activity; a similar effect was also seen with serum albumin. In silico docking studies with PPAP53 revealed that it binds to the F1 pocket of human serum albumin with a binding energy of −7.5 kcal/mol. In an infection model of septic arthritis, PPAP23 decreased the formation of abscesses and S. aureus load in kidneys; in a mouse skin abscess model, topical treatment with PPAP53 reduced S. aureus counts. Both PPAPs were active against anaerobic Gram-positive gut bacteria such as neurotransmitter-producing Clostridium, Enterococcus or Ruminococcus species. Based on these results, we foresee possible applications in the decolonization of pathogens.

Before the emergence of SARS-CoV-2, tuberculosis (TB) was the leading cause of infectious disease mortality worldwide. Like all antibiotic-exposed bacteria Mycobacterium tuberculosis (Mtb) developed multidrug-resistant (MDR) and exten-sively drug-resistant (XDR) strains which require new antibiotics with novel mechanism of actions. Hyperforin, a natural type-A polyprenylated polycyclic acylphloroglucinol (PPAP) isolated from St. John’s wort, is known for its antibacterial, antidepressant and antimycobacterial activity. However, hyperforin is not stable and easily degradable in light, heat it and oxidizes. Here we report photo- and benchstable type-B PPAPs with structural similarity to hyperforin and enhanced an-timycobacterial activity. We tested a panel of PPAPs and identified our previously reported molecule PPAP22 as lead compound. Converting PPAP22 into the corresponding sodium salt, PPAP53, enhanced the solubility dramatically. We show that PPAP53 inhibits the growth of virulent, extracellular Mtb. Strikingly, the activity is more pronounced intracel-lular Mtb residing in human primary macrophages without damaging the host cell or lung cells. Importantly PPAP53 was also highly active against drug-resistant Mtb. Additionally, we analysed the in vitro properties of PPAP53 in terms of CYP-induction and PXR interaction. Taken together we introduce type-B PPAPs are a new class of antimycobacterial com-pounds, with remarkable activity and favorouble physical properties.

The selective modulation of TRPC6 ion channels has emerged as a promising therapeutic approach for treating neuro-degenerative diseases and depression. Here, we present a significant advancement in this field by demonstrating the se-lective activation of TRPC6 using a metallated type-B PPAP, designated as PPAP53. The success of PPAP53 is attributed to the utilization of the 1,3-diketone motif present in PPAPs for metal coordination. The metallated PPAPs exhibit water solubility and equipotent activity compared to hyperforin, which is a natural product and considered the gold standard in the field. Notably, and in sharp contrast to type-A PPAPs, type-B PPAPs possess unique properties such as synthetic ac-cessibility in gram scale, facile derivatization, being thermally stable and stable against photochemical oxidation. Our detailed investigations reveal that PPAP53 selectively binds to the C-terminus of TRPC6. Although cryo electron micros-copy has resolved the majority of the TRPC6 structure, the binding site in the C-terminus remained unresolved. To ad-dress this issue, we employed state-of-the-art artificial intelligence-based protein structure prediction algorithms, includ-ing AlphaFold2, ColabFold, and trRosetta, to predict the missing C-terminus region. Our computational results, validated against experimental data, indicate that PPAP53 binds to the 777LLKL780-region of the C-terminus, thus providing critical insights into the binding mechanism of PPAP53 with TRPC6.