Overcoming drug-resistance and the subsequent relapse that often occurs with monotherapy is crucial in the treatment of acute myeloid leukemia. We here demonstrate that therapy-resistant leukemia initiating cells can be targeted using a novel inhibitor of mitochondrial transcription (IMT). The compound inhibits mitochondrial RNA polymerase activity and sensitizes the resistant population to the induction of apoptosis. In vitro studies on acute myeloid leukemia cells demonstrate that IMT prevents cell proliferation, and together with a selective BCL-2 inhibitor, venetoclax, induces apoptosis and suppress oxidative phosphorylation (OXPHOS) synergistically. AML mouse models treated with IMT in combination with venetoclax show prolonged survival in venetoclax-resistant models. Our findings suggest that certain therapy-resistant leukemia cell populations display a unique dependency on mitochondrial transcription and can be targeted with IMT.

Abstract The introduction of targeted immunomodulating agents has transformed cancer treatment over the last decade by demonstrating unprecedented efficacy in patients who respond. However, limited clinical response rates as well as adverse events underline the need to identify additional novel modalities in cancer immunotherapy. Here we report on the discovery of a novel class of low molecular weight compounds for oral application that selectively enhance tumor-reactive T cell cytotoxicity. Hit-to-lead development of compound hit series A was performed based on medicinal chemistry to investigate structure-activity-relations. A-306, a representative of the A compound class, induced increased T cell proliferation, elevated levels of the activation markers CD25 and CD69 as well as enhanced secretion of IFN-γ, IL-2 and TNF-α upon anti-CD3/CD28 stimulation. A-306 treatment without T cell receptor ligation had no effect on T cell activation, suggesting selective activation potential only in the context of antigen encounter. Furthermore, A-306 strengthened T-cell mediated killing of M21 melanoma cells and enhanced T cell function in response to viral antigens by showing a Th1 signature. Medicinal chemistry efforts resulted in front runner compounds A-481 and A-687 that show a similar profile and potencies down to nanomolar level. In a murine B16-SIY melanoma and EO771 breast cancer model oral single-agent administration with front runner compounds was well tolerated, showed good bioavailability in lymphoid organs and plasma and resulted in significant tumor growth inhibition beyond the end of treatment (D21). T cells from tumor draining lymph nodes of long-term surviving mice in both cancer models showed a distinct activation pattern indicative of anti-tumor immunity. Furthermore, oral application of A-481 in combination with intra peritoneal administration of anti-PD1 antibodies eradicated B16-SIY melanoma cells in a synergistic manner. Taken together, we report on the identification of a novel class of small molecules possessing high potential for selective anti-tumor activation of the immune system upon oral administration. Front runner compounds A-481 and A-687, which significantly inhibit tumor growth in a B16-SIY melanoma and EO771 breast cancer mouse model, lead to prolonged survival beyond the end of treatment and display a good safety profile. The final candidate compound will undergo testing in toxicity studies and enter a first-in-human trial in selected solid tumors in 2025. Citation Format: Bianca Gapp, Gunther Zischinsky, Maria Urban, Nora Birnbacher, Alice Schwarzböck, Sabine Grünaug, Tommaso Gastaldi, Anton Stütz, Peter Nussbaumer, Stefan Stranner, Julia Buchberger, Andreas Birbach, Daniela Schögl, Mario Kuttke, Alexander Dohnal, Romana Gugenberger. A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5936.

Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.

The European Lead Factory combines assets and experience from major pharma with innovation and agility of academia and SMEs in a collaborative platform to expand access to high-throughput screening. With many successes heading towards the clinic, the organization is broadening its approach to screening and partnering. The European Lead Factory combines assets and experience from major pharma with innovation and agility of academia and SMEs in a collaborative platform to expand access to high-throughput screening. With many successes heading towards the clinic, the organization is broadening its approach to screening and partnering.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system 1 – 6 . The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS 7 – 17 , and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

Due to the distinct potential applications of squalene epoxidase inhibitors – treatment of fungal infections by selective blocking of ergosterol biosynthesis in fungi, or lowering of cholesterol levels by inhibition of human sterol biosynthesis — differential biological profiles of the drugs and, hence, different test systems were required for the respective indication. Due to the existence of highly active and selective fungal squalene epoxidase inhibitors (the allylamine, the homopropargylamine, the benzylamine, and the thiocarbamate antimycotics), squalene analogues have been of no importance as inhibitors of the fungal enzyme. Allylamine derivatives constitute the most important class of squalene epoxidase inhibitors. A first group of compounds is highly selective for the fungal enzyme and used safely and successfully in the topical and systemic therapy of mycoses. A second group selectively inhibits mammalian squalene epoxidase, and appears to have a promising potential for the treatment of hypercholesterolemia.

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway sigma E represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.

G-Protein Coupled Receptors (GPCR) are involved in all the major signaling pathways. As a result, they often serve as potential target for therapeutic drugs. In this study we analyze publicly available assays involving different classes of GPCR to identify false positives. Using the latest developments in Machine Learning, we then build models that can predict such compounds with high confidence. Given the ubiquity of GPCR assays, we believe such models will be very helpful in flagging potential false positives for further testing.

Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3β is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3β. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3β in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.

Covalent modifications of nonactive site lysine residues by small molecule probes has recently evolved into an important strategy for interrogating biological systems. Here, we report the discovery of a class of bioreactive compounds that covalently modify lysine residues in DegS, the rate limiting protease of the essential bacterial outer membrane stress response pathway. These modifications lead to an allosteric activation and allow the identification of novel residues involved in the allosteric activation circuit. These findings were validated by structural analyses via X-ray crystallography and cell-based reporter systems. We anticipate that our findings are not only relevant for a deeper understanding of the structural basis of allosteric activation in DegS and other HtrA serine proteases but also pinpoint an alternative use of covalent small molecules for probing essential biochemical mechanisms.

The ups and downs of a multi-year medicinal chemistry optimisation effort in the quest of identifying inhibitors of human steroid sulfatase as potential topical therapeutics are summarised. The focus lies on dead ends, redirection and mastering of the hurdles rather than detailed structure–activity relationship results of a series. In addition to the usual multi-parameter optimisation towards drug-like properties, unexpected barriers prohibitive for the planned clinical application had to be overcome. While a substrate- and mechanism-based design approach rapidly yielded aryl sulfamates as potent, irreversible enzyme inhibitors, this inhibitor class had to be abandoned due to inherent chemical instability in solution. The rescue of the project was a hit class from a random screening approach, but from there it still took quite some efforts to invent potent reversible inhibitors appropriate for development towards a clinical candidate.

The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.

AbstractEine Reaktion auf den Strukturwandel in der Arzneimittelforschung ist das vor zehn Jahren gegründete Unternehmen Lead Discovery Center. Es vermittelt zwischen Industrie und Akademia.

(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg-1 i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation.

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

The K-Ras GTPase is a major target in anticancer drug discovery. However, direct interference with signaling by K-Ras has not led to clinically useful drugs yet. Correct localization and signaling by farnesylated K-Ras is regulated by the prenyl binding protein PDEδ. Interfering with binding of PDEδ to K-Ras by means of small molecules provides a novel opportunity to suppress oncogenic signaling. Here we describe the identification and structure-guided development of novel K-Ras-PDEδ inhibitor chemotypes based on pyrrolopyridazinones and pyrazolopyridazinones that bind to the farnesyl binding pocket of PDEδ with low nanomolar affinity. We delineate the structure-property relationship and in vivo pharmacokinetic (PK) and toxicokinetic (Tox) studies for pyrazolopyridazinone-based K-Ras-PDEδ inhibitors. These findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic Ras.

The sesquiterpene (-) Englerin A (EA) is an organic compound from the plant Phyllanthus engleri which acts via heteromeric TRPC4/C1 channels to cause cytotoxicity in some types of cancer cell but not normal cells. Here we identified selective cytotoxicity of EA in human synovial sarcoma cells (SW982 cells) and investigated the mechanism. EA induced cation channel current (Icat) in SW982 cells with biophysical characteristics of heteromeric TRPC4/C1 channels. Inhibitors of homomeric TRPC4 channels were weak inhibitors of the Icat and EA-induced cytotoxicity whereas a potent inhibitor of TRPC4/C1 channels (Pico145) strongly inhibited Icat and cytotoxicity. Depletion of TRPC1 converted Icat into a current with biophysical and pharmacological properties of homomeric TRPC4 channels and depletion of TRPC1 or TRPC4 suppressed the cytotoxicity of EA. A Na+/K+-ATPase inhibitor (ouabain) potentiated EA-induced cytotoxicity and direct Na+ loading by gramicidin-A caused Pico145-resistant cytotoxicity in the absence of EA. We conclude that EA has a potent cytotoxic effect on human synovial sarcoma cells which is mediated by heteromeric TRPC4/C1 channels and Na+ loading.

Selective inhibition of exclusively transcription-regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY-958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.