Slava Ziegler

We report the design, synthesis, and biological characterization of a pseudo‐NP (PNP) collection inspired by the mesembrine alkaloid scaffold. A scalable, microwave‐assisted intramolecular Diels–Alder furan cyclization enabled efficient access to the 3a‐aryloctahydroindole (AOHI) core, which served as a versatile intermediate for diversification through NP‐fragment fusion. The resulting AOHI‐based PNPs explore chemical space bridging properties of NPs and drug‐like molecules. Morphological and bioenergetic profiling revealed distinct mitochondrial phenotypes, including altered morphological features and respiration consistent with complex I perturbation. These findings highlight the AOHI scaffold as a versatile and synthetically accessible alkaloid‐derived fragment for PNP design and as a valuable source for novel biologically active compound classes.

Targeted protein degradation modulates protein function beyond the inhibition of enzyme activity or protein–protein interactions. Most degrader drugs function by directly mediating the proximity between a neosubstrate and a hijacked E3 ligase. Here we identify pseudo-natural products derived from (−)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2 ^KLHDC3 , which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce the formation of kynurenine by both inhibition and induced degradation of the enzyme and thus also modulate the non-enzymatic functions of IDO1. This unique mechanism of action may open up alternative therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

For targeted covalent modification at low-reactivity carboxylates with biocompatible electrophiles, new approaches are in high demand. Engineering of the HaloTag protein facilitates such a covalent reaction between chloroalkanes and an aspartate residue. We demonstrate that conversely, engineering stable ligands can also enable covalent targeting of an acid residue in a protein binding site. Using the chaperone PDEδ, which shuttles lipidated oncoproteins and thereby mediates their signaling activity, we show that equipping noncovalent inhibitors with a benzyl fluoride-based electrophile leads to covalent modification of a specific glutamate p.E88 in the ligand binding site. The best inhibitor, Deltafluorine, embodies a 3-fluoromethyl-pyridyl group and is stable to nucleophiles like glutathione, phosphate, acetate, and citrate. In cells, Deltafluorine combines noncovalent and covalent reactivity to demonstrate distinct cellular profiles and inhibits signaling through the MAP-kinase and Akt-mTOR pathways. In an autochthonous mouse model of highly aggressive Kras^G12D-driven lung adenocarcinoma, Deltafluorine treatment significantly reduces tumor volume.

We report the design, synthesis, and biological char-acterization of a pseudo-NP (PNP) collection in-spired by the mesembrine alkaloid scaffold. A scala-ble, microwave-assisted intramolecular Diels-Alder furan cyclization enabled efficient access to the 3a-aryloctahydroindole (AOHI) core, which served as a versatile intermediate for diversification through NP-fragment fusion. The resulting AOHI-based PNPs explore chemical space bridging properties of NPs and drug-like molecules. Morphological and bioen-ergetic profiling revealed distinct mitochondrial phe-notypes, including altered morphological features and respiration consistent with complex I perturba-tion. Seahorse flux analysis showed that fusion with a pyridine robustly modulated mitochondrial function, indicating an upstream effect of complex V. Taken together, these findings highlight the AOHI scaffold as a versatile and synthetically accessible alkaloid-derived fragment for pseudo-natural product design and as a valuable source for novel biologically active compound classes.

Terpenoid alkaloids are derived from the fusion of structurally diverse terpenoid‐ and alkaloid moieties. The biologically relevant chemical space defined by this unique natural product (NP) class may be explored beyond the limitations of biosynthetic pathways by means of the pseudo natural product (PNP) principle, i.e., by combination of NP fragments in different arrangements. We describe the design, synthesis and structural evolution of a monoterpene–pyrrolidine PNP collection obtained by functionalization and combination of bicyclic monoterpenes with pyrrolidine alkaloid‐derived fragments. Diverse fusion strategies led to the discovery of (‐)‐myrtenal‐pyrrolidine PNPs that are indoleamine‐2,3‐dioxygenase 1 (IDO1) inhibitors and degraders, termed iDegs. Structural fine‐tuning modulated both degradation and inhibition potencies. Co‐crystallization revealed that iDegs induce unprecedented changes in the C‐terminus of IDO1 which promote degradation. iDegs inhibited tumor growth in SKOV‐3 tumor‐bearing mice and led to prolonged survival, which promises to inspire novel medicinal chemistry programs aimed at IDO1 in different diseases.

Terpenoid alkaloids are derived from the fusion of structurally diverse terpenoid‐ and alkaloid moieties. The biologically relevant chemical space defined by this unique natural product (NP) class may be explored beyond the limitations of biosynthetic pathways by means of the pseudo natural product (PNP) principle, i.e., by combination of NP fragments in different arrangements. We describe the design, synthesis and structural evolution of a monoterpene–pyrrolidine PNP collection obtained by functionalization and combination of bicyclic monoterpenes with pyrrolidine alkaloid‐derived fragments. Diverse fusion strategies led to the discovery of (‐)‐myrtenal‐pyrrolidine PNPs that are indoleamine‐2,3‐dioxygenase 1 (IDO1) inhibitors and degraders, termed iDegs. Structural fine‐tuning modulated both degradation and inhibition potencies. Co‐crystallization revealed that iDegs induce unprecedented changes in the C‐terminus of IDO1 which promote degradation. iDegs inhibited tumor growth in SKOV‐3 tumor‐bearing mice and led to prolonged survival, which promises to inspire novel medicinal chemistry programs aimed at IDO1 in different diseases.

In pseudo‐natural product (PNP) design, natural product fragments are combined in arrangements not found in nature to afford novel biologically relevant NP‐related compound classes. A pseudo‐natural product compound collection inspired by aspidospermidine‐, strychnos‐, and koumine monoterpene alkaloids was designed and rapidly synthesized to afford a collection of 78 compounds representing eight structurally different PNP classes. Evaluation of the collection for biological relevance by means of the cell painting assay, including a recently reported subprofile analysis, revealed that one of the structural classes is enriched in and defines a new chemotype of de novo pyrimidine biosynthesis inhibitors. Our results exemplify the combination of pseudo‐natural product design and unbiased morphological screening technologies as an efficient means to rapidly access and identify novel biologically relevant chemotypes.

The indole moiety is a privileged fragment that frequently populates existing bioactive compound collections. The development of an indole‐dearomatization sequence and its application for library expansion of a collection of indole‐containing pseudo‐natural products (NPs) are described. The resulting compounds are topologically distinct from the original compound class. Phenotyping by means of the cell painting assay initially indicates that the dearomatized compounds are morphologically different than the original pseudo‐NP compound class and guiding NPs. However, analysis by means of a new subprofile analysis of the same cell painting assay data indicates that similar morphologies persist throughout the compound classes. Further biological studies support the findings of the subprofile analysis and highlight its potential to more effectively characterize novel compounds. The biological findings suggest that a plethora of indole‐dearomatization reactions can be applied to existing indole‐containing compound collections to rapidly access new biologically relevant scaffolds.

The tumor microenvironment (TME) is a pro‐cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer‐supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor β (TGFβ). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell‐mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell‐mediated cytotoxicity, we developed a phenotypic co‐culture assay of cancer cells and primary lymphocytes suitable for medium‐throughput screening. We discovered small molecules that restore NK cell‐mediated cytotoxicity through diverse mechanisms. The potent TGFβ type I receptor (TGFβR‐1) inhibitor, RepSox, stood out as superior to other TGFβR‐1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGFβR‐1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity.

Summary: The tumor microenvironment (TME) is a pro-cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer-supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor β (TGFβ). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell-mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell-mediated cytotoxicity, we developed a phenotypic co-culture assay of cancer cells and primary lymphocytes suitable for medium-throughput screening. We discovered small molecules that restore NK cell-mediated cytotoxicity through diverse mechanisms. The potent TGFβ type I receptor (TGFβR-1) inhibitor, RepSox, stood out as superior to other TGFβR-1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGFβR-1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity.