Thomas Lundbäck

The sterile alpha and TIR motif containing 1 (SARM1) enzyme is a key driver of axonal degeneration in response to injury, making it an attractive target for treating chemotherapy-induced peripheral neuropathy (CIPN) and other nervous system diseases. In this study, we identified and optimised a class of base-exchange inhibitors (BEXi) targeting human SARM1 and explored their molecular interactions and conformational effects using cryo-EM, HDX-MS and SAXS. Although BEXi produced robust inhibition across all biochemical and cellular assay formats, application at sub-inhibitory concentrations consistently led to paradoxical SARM1 activation, and in neuronal assays, accelerated neurite degeneration. Further analysis showed that BEXi only delayed, rather than prevented, neurite degeneration when applied to primary neuronal cells, even at exceedingly high inhibitor concentrations. These results prompted us to discontinue BEXi development in favour of alternative strategies, underscoring the complexity of SARM1 as a therapeutic target and the need for comprehensive, mechanistically informed screening cascades.

Human loss-of-function variants in 17βHSD13 have been associated with reduced risk and progression of metabolic and alcohol-related liver disease. Although in vitro experiments implicate 17βHSD13 in the processing of steroidal and lipogenic substrates, establishing an unambiguous mechanistic link to disease causation remains challenging, and the absence of robust pharmacodynamic biomarkers complicates clinical dose selection based on tissue target occupancy. To address this, we implemented a medium-throughput cellular thermal shift assay (CETSA) in humanized mouse liver homogenates to quantify the binding of BI-3231, an uncompetitive 17βHSD13 inhibitor, and compared results with functional cell assays. We systematically profiled isothermal dose-response fingerprints (ITDRF_CETSA) across 58-70.5 °C to account for temperature-induced relaxation of binding equilibria at elevated temperatures, which produced pronounced right-shifts in the apparent potency and confirmed NAD⁺-dependent binding. Complementary surface plasmon resonance (SPR) measurements across 5-42 °C defined the temperature dependence of NAD⁺ and BI-3231 binding. Extrapolating SPR affinities to the CETSA temperature range showed convergence with ITDRF_CETSA fingerprints, supporting the use of the SPR-derived K_D for BI-3231 (2.5 nM at 37 °C; pK_D = 8.60 ± 0.07, 95% CI) to estimate the occupancy also in liver homogenates. This work provides a generalizable approach to quantify target engagement for CETSA-responsive drug targets, while underscoring that occupancy estimates for uncompetitive inhibitors must incorporate cofactor saturation.

The sterile alpha and TIR motif containing 1 (SARM1) enzyme is a key driver of axonal degeneration in response to injury, making it an attractive target for treating chemotherapy-induced peripheral neuropathy (CIPN) and other nervous system diseases. In this study, we identified and optimised a new class of base-exchange inhibitors (BEXi) targeting SARM1 and explored their molecular interactions and conformational effects using cryo-EM, HDX-MS and SAXS. Although BEXi produced robust inhibition across all biochemical and cellular assay formats, application at sub-inhibitory concentrations consistently led to paradoxical SARM1 activation, and in neuronal assays, accelerated neurite degeneration. Further analysis showed that BEXi only delayed, rather than prevented, neurite degeneration when applied to primary neuronal cells, even at exceedingly high inhibitor concentrations. These results prompted us to discontinue BEXi development in favour of alternative strategies, underscoring the complexity of SARM1 as a therapeutic target and the need for comprehensive, mechanistically informed screening cascades.

The disruption of microtubule dynamics serves as a pivotal strategy for eliminating tumor cells, despite its accompanying toxicities affecting non‐tumor cells. This study investigates the potential of selectively targeting γ‐tubulin1 (TUBG1) as a therapeutic strategy in cancer treatment. By elucidating the TUBG1–E2F1–retinoblastoma protein (RB1) network, we introduce a novel compound, 4‐(6‐((3‐Methoxyphenyl)amino)pyrimidin‐4‐yl)‐N,N‐dimethylbenzenamine, (L12). L12 treatment enhanced RB1 expression and selectively targeted cells with impaired RB1 signaling, while reduced E2F1 expression attenuated its cytotoxicity. Furthermore, L12‐mediated cytotoxicity depends on an E2F1‐mediated upregulation of procaspase 3 expression, highlighting the role of E2F1 in the apoptotic response. Unlike traditional tubulin‐targeting agents, L12's specificity for tumor cells lies in its inhibitory effects on TUBG1, without affecting the second human isoform of TUBGs, TUBG2. Despite its interaction with specific kinases, the concentrations required for antitumor effects are 100‐fold lower than those influencing kinase activities. Subsequent investigations underscore L12's reduced neuronal axonal toxicity compared to vincristine. Lastly, L12 demonstrates promising results in inhibiting tumor growth in xenografted small cell lung cancer models, demonstrating potential specificity toward tumor cells while minimizing adverse effects on healthy tissues. This research emphasizes the potential of TUBG1 inhibitors as a promising advancement in personalized chemotherapy approaches and their potential as a groundbreaking treatment for various cancers.

The drug development landscape is expanding to include drug modalities such as PROteolysis-TArgeting Chimeras (PROTACs) and peptides, offering possibilities for previously intractable biological targets. However, with their size and chemical nature, they diverge from established frameworks for the prediction of oral bioavailability. This evolution to larger and more complex molecules necessitates new methodologies and prediction models to continuously expand on bioavailability guidelines. We describe the high-capacity adoption of two chromatographic physicochemical assays and their application for iterative compound optimization to achieve oral bioavailability. We further describe how these data underpin the continuous refinement of internal machine learning models, which guide compound synthesis decisions in the molecular design phase. Based on data for a set of 691 PROTACs, and two project examples, we confirm a sweet spot for oral bioavailability at log D values higher than the norm for small molecules and show how experimental data and prediction models synergize to effectively drive chemistry optimization.

An all-photonic method is described, in which (i) the release of an active kinase inhibitor is controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the inhibitor to its corresponding target.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC ₅₀ of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC₅₀ value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound’s metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn²⁺ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Precise length, shape, and linker attachment points are all integral components to designing efficacious proteolysis targeting chimeras (PROTACs). Due to the synthetic complexity of these heterobifunctional degraders and the difficulty of computational modeling to aid PROTAC design, the exploration of structure-activity relationships remains mostly empirical, which requires a significant investment of time and resources. To facilitate rapid hit finding, we developed capabilities for PROTAC parallel synthesis and purification by harnessing an array of preformed E3-ligand-linker intermediates. In the next iteration of this approach, we developed a rapid, nanomole-scale PROTAC synthesis methodology using amide coupling that enables direct screening of nonpurified reaction mixtures in cell-based degradation assays, as well as logD and EPSA measurements. This approach greatly expands and accelerates PROTAC SAR exploration (5 days instead of several weeks) as well as avoids laborious and solvent-demanding purification of the reaction mixtures, thus making it an economical and more sustainable methodology for PROTAC hit finding.