Biomedical subjects

Stefan Knapp

Publications and source records attributed to Stefan Knapp.

15 recordsLinked to original sources

PubMed2026

Cell type-selective targeting by heterobifunctional protein binders via in-cell enrichment.

Non-catalytic heterobifunctional protein binders promise to expand the range of therapeutic options by establishing complexes between key target proteins and accessory presenter proteins equipped with additional properties. Here, we systematically investigate the rational design of such molecules, explore the biochemical basis of complex formation and determine how they achieve cellular efficacy using the endogenously expressed immunophilin FKBP12 as presenter protein and the transcriptional regulator BRD4 as target protein. We present classes of bifunctional molecules that enable selective, FKBP12-dependent killing of specific cell types at subnanomolar concentrations and allow to differentiate between closely related bromodomains of the BET family. We propose that the strongly potentiated efficacy of these bifunctional compounds is based on cellular enrichment through binding to the highly abundant presenter protein FKBP12, a mechanism we term "CellTrap". Our findings substantiate the concept that highly expressed, non-essential proteins can be repurposed as selective recruiters to expand therapeutic windows of existing small-molecule inhibitors, opening new avenues for designing targeted drugs with improved cell-type specificity.

Tacrolimus Binding Protein 1A↗

PubMed2026

Selective Macrocyclic WEE1 Kinase Inhibitors with Strong Efficacy against Patient-Derived Colorectal Cancer Organoids.

Macrocyclization can enhance the selectivity of acyclic compounds toward structurally similar biological targets such as kinases. WEE1 regulates cellular homeostasis and is a promising target in oncology. The clinical candidate AZD1775 (1) failed to progress past Phase II trials because of patient tolerability issues, likely due to off-target inhibition of polo-like kinase 1 (PLK1). Herein, a computer-aided drug design approach was conducted to develop a macrocycle based on the 1-WEE1 X-ray cocrystal structure. Significantly enhanced WEE1 inhibitory selectivity over PLK1 was determined for leading macrocycle 2, which also demonstrated broader kinome-wide selectivity. Patient-derived organoids from colorectal cancer (CRC) peritoneal and liver metastases, treated with 2, demonstrated comparably strong or enhanced anticancer efficacy compared to that of 1. Against patient-matched normal colon vs primary CRC organoids, 2 potently and selectively treated CRC, as well as enhanced DNA damage compared to 1. Finally, the X-ray cocrystal structure of 2 bound to WEE1 validated its computationally predicted bioactive binding mode.

Humans↗

PubMed2026

Macrocyclization of Broad-Spectrum Kinase Inhibitor Bosutinib Leads to Potent and Selective Quinoline-Based HIPK4 Inhibitor AZ137.

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest its involvement in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. These investigations are currently hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50: 11 nM; cellular EC50: 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.

Quinolines↗

PubMed2026

Accelerate Your Science: Direct-to-Biology Strategies in Medicinal Chemistry.

Direct-to-biology (D2B) is a powerful strategy that accelerates early drug discovery. It enables compounds to be synthesized in miniaturized formats and evaluated directly as crude reaction mixtures. This bypasses the need for purification during the initial design-make-test cycle. Advances in robust synthetic methodologies, automation, reaction miniaturization, and biological screening have transformed D2B from a proof-of-concept approach into a versatile medicinal chemistry platform. This platform is applicable to fragment optimization, covalent ligands, macrocycles, proteolysis-targeting chimeras (PROTACs), molecular glues, and cellular phenotypic screening. This perspective focuses on the synthetic transformations, assay technologies, and platform implementations that drive modern D2B workflows. It emphasizes reaction robustness, assay compatibility, and practical implementation. Analysis of the current literature revealed that D2B is more governed by reaction reliability than synthetic diversity. Amide coupling and click chemistry dominate reported workflows, while more complex transformations remain underexplored. We discuss the complementary strengths and limitations of biochemical, biophysical, and cellular readouts, identify current bottlenecks in reaction scope and data management, and highlight emerging opportunities arising from reaction miniaturization, machine learning, automated experimentation, and advanced synthetic methodologies. Rather than replacing conventional medicinal chemistry, D2B fundamentally shifts experimental effort from purification toward early biological validation and is poised to become an integral component of future medicinal chemistry workflows.

Humans↗

PubMed2026

A targetable dependency on nonsense-mediated decay for cellular homeostasis and immune control in small cell lung cancer.

Small cell lung cancer (SCLC) is one of the most aggressive malignancies, characterized by rapid metastatic dissemination and poor overall survival. Despite harboring excessive alterations, expectedly resulting in immunogenic neoantigens, patients with SCLC remain largely refractory to immunotherapy. We found abundant frameshift mutations in SCLC, regarded as highly immunogenic, counterbalanced by a hyperactive nonsense-mediated decay (NMD) pathway, responsible for frameshift-mRNA degradation. NMD activity correlated with tumor mutational burden (TMB) across cancers, suggesting that SCLC and other TMBhigh cancers may depend on NMD to limit the accumulation of mutation-derived byproducts in order to maintain cellular homeostasis and evade immune recognition. In TMBhigh SCLC models, inhibition of NMD impaired cell proliferation and induced ER stress-dependent apoptosis due to the accumulation of misfolded proteins. Genetic and pharmacological NMD inhibition in vivo effectively controlled TMBhigh tumor growth without overt toxicity. By integrating genome and transcriptome sequencing with MHC-I immunopeptidomics and functional in vitro and in vivo assays, we identified that NMD inhibition boosted neoantigen expression and presentation by tumor cells and increased T cell recognition, thus enhancing overall tumor immunogenicity and further improving immunotherapy efficacy in vivo. Our work shows that SCLC - as a TMBhigh cancer - relies on NMD for survival and immune escape, uncovering a novel TMB-dependent tractable vulnerability for this devastating disease.

Humans↗

PubMed2026

Macrocyclization of Broad-Spectrum Kinase Inhibitor Bosutinib leads to Potent and Selective Quinoline-based HIPK4 Inhibitor AZ137.

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.

Journal Article↗

PubMed2025

Probing the Protein Kinases' Cysteinome by Covalent Fragments.

Protein kinases are important drug targets, yet specific inhibitors have been developed for only a fraction of the more than 500 human kinases. A major challenge in designing inhibitors for highly related kinases is selectivity. Unlike their non-covalent counterparts, covalent inhibitors offer the advantage of selectively targeting structurally similar kinases by modifying specific protein side chains, particularly non-conserved cysteines. Previously, covalent fragment screens yielded potent and selective inhibitors for individual kinases such as ERK1/2 but have not been applied to the broader kinome. Furthermore, many of the accessible cysteine positions have not been addressed so far. Here, we outline a generalizable approach to sample ATP-site cysteines with fragment-like covalent inhibitors. We present the development of a kinase-focused covalent fragment library and its systematic screening against a curated selection of 47 kinases, with 60 active site-proximal cysteines using LC/MS and differential scanning fluorimetry (DSF) assays, followed by hit validation through various complementary techniques. Our findings expand the repertoire of targetable cysteines within protein kinases, provide insight into unique binding modes identified from crystal structures and deliver isoform-specific hits with promising profiles as starting points for the development of highly potent and selective covalent inhibitors.

Protein Kinase Inhibitors↗

PubMed2003

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Amino Acid Sequence↗

PubMed2003

Protein stabilisation by compatible solutes: effect of mannosylglycerate on unfolding thermodynamics and activity of ribonuclease A.

Differential scanning calorimetry, optical spectroscopy, and activity measurements were used to investigate the effect of mannosylglycerate, a negatively charged osmolyte widely distributed among thermophilic and hyperthermophilic archaea and bacteria, on the thermal unfolding of ribonuclease A (RNase A). For comparison, assays in the presence of trehalose, a canonical solute in mesophiles, and potassium chloride were also carried out. A thermodynamic analysis was performed by using differential scanning calorimetry data. The changes in the heat capacity for unfolding were similar for the different solutes examined. Mannosylglycerate was an efficient thermostabiliser of RNase A and induced an increase of 6 degrees C mole(-1) in the melting temperature. Moreover, the performance of mannosylglycerate as a stabiliser depended on the net charge of the molecule, with the maximal effect being observed at pH values above 4.5. Analysis of the enthalpic and entropic contributions to unfolding, derived from calorimetric data, revealed that the stabilisation rendered by mannosylglycerate is primarily achieved through a decrease in the unfolding entropy. Also, the number of protons taken up by RNase A upon denaturation in the presence of mannosylglycerate was considerably higher than with other solutes, a result consistent with a more rigid structure of the native protein. Mannosylglycerate (potassium salt) inhibited the activity of RNase A, albeit to a smaller extent than KCl, and acted as an efficient suppressor of aggregation of the denatured protein, thereby having a remarkable beneficial effect on the inactivation of RNase A upon thermal denaturation. The results are discussed in view of the physiological role of this charged compatible solute.

Animals↗

PubMed2003

Hot spots in Tcf4 for the interaction with beta-catenin.

The interaction of beta-catenin with T-cell factor (Tcf) 4 plays a central role in the Wnt signaling pathway and has been discussed as a possible site of intervention for the development of anti-cancer drugs. In this study, we performed Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface and studied the binding energetics of these mutants using isothermal titration calorimetry. Binding of Tcf4 was found to be highly cooperative. Single site mutations of most Tcf4 residues resulted in a significant reduction in binding enthalpies but in similar binding constants as compared with wild type Tcf4. Interestingly, this was also true for residues that are disordered in the reported crystal structures. The mutation D16A caused the largest reduction in binding constant (50-fold) accompanied by a large unfavorable enthalpy change (DeltaDeltaHobs) of +8 kcal/mol at 25 degrees C. In contrast, the mutation of the Tcf residues Glu24 and Glu28, which have been proposed as an interaction hot spot due to their location in a field of strong positive electrostatic potential on the beta-catenin surface (charge button), resulted only in a significant reduction of binding enthalpies, which were largely compensated for by unfavorable entropic contributions to the binding. Other mutations that significantly reduced Tcf binding constants were D11A and alanine mutations of the hydrophobic residues Leu41, Val44, and Leu48. The measured thermodynamic data are discussed with the available structural information of Tcf-beta-catenin crystal structures and allow us to propose possible sites for development of Tcf antagonists.

Amino Acid Sequence↗

PubMed2003

Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules.

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.

Animals↗

PubMed2002

High-throughput NMR-based screening with competition binding experiments.

The Achilles heel of ligand-based NMR screening methods is their failure to detect high-affinity ligands and molecules that bind covalently to the receptor. We have developed a novel approach for performing high-throughput screening with NMR spectroscopy that overcomes this limitation. The method also permits detection of potential high-affinity molecules that are only marginally soluble, thus significantly enlarging the diversity of compounds amenable to NMR screening. The techniques developed utilize transverse and/or selective longitudinal relaxation parameters in combination with competition binding experiments. Mathematical expressions are derived for proper setup of the NMR experiments and for extracting an approximate value of the binding constant for the identified ligand from a single-point measurement. With this approach it is possible to screen thousands of compounds in a short period of time against protein or DNA and RNA fragments. The methodology can also be applied for screening plant and fungi extracts.

Binding, Competitive↗

PubMed2002

NMR-Based screening with competition water-ligand observed via gradient spectroscopy experiments: detection of high-affinity ligands.

Water-ligand observed via gradient spectroscopy (WaterLOGSY) represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. The method is useful for the detection of compounds binding to a receptor with binding affinity in the micromolar range. The Achille's heel of the method, as with all the techniques that detect the ligand resonances, is its inability to identify strong ligands with slow dissociation rates. We show here that the use of a reference compound with a known K(D) in the micromolar range together with properly designed competition binding experiments (c-WaterLOGSY) permits the detection of strong binders. A derived mathematical expression is used for the selection of the appropriate setup NMR experimental conditions and for an approximate determination of the binding constant. The experiment requires low ligand concentration, therefore allowing its application in the identification of potential strong inhibitors that are only marginally soluble. The technique is particularly suitable for rapid screening of chemical mixtures and plant or fungi extracts.

Algorithms↗

PubMed2002

Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint.

The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2-Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1-Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular 'safety belts'. We show that Mad1 is a competitive inhibitor of the Mad2-Cdc20 complex, and propose that the Mad1-Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1-Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1-Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1-Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the 'safety belt' mechanism may prolong the lifetime of Mad2-ligand complexes.

Amino Acid Sequence↗

PubMed2002

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases.

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.

Amino Acid Sequence↗