The Akt family of serine/threonine kinases plays a crucial role in various cellular processes, including proliferation, survival, and metabolism. Three Akt isoforms (Akt1, Akt2, and Akt3) have distinct physiological roles, and while individual isoform dysregulation is disease-linked, unselective Akt inhibition leads to side effects. Here, we report the development of selective covalent-allosteric Akt inhibitors (CAAIs) targeting Akt2 and Akt3 while sparing Akt1. Guided by protein x-ray crystallography and molecular modeling, key structural differences within the allosteric pockets of the isoforms were identified and exploited in a structure-based design strategy. By stabilizing the inactive kinase conformation, CAAIs overcome the intrinsic selectivity limitations of ATP-competitive inhibitors. After biological characterization, the pyrazole-containing inhibitors emerged as the most potent and selective Akt2 inhibitors, while inhibitors with pyridines as an isoform-selective element were predominantly targeting Akt3 selectively. Importantly, these new inhibitors were also evaluated in patient-derived colorectal cancer organoids. Co-crystal structures of inhibitors bound to an engineered construct mimicking the Akt2 allosteric pocket elucidated the molecular basis of isoform selectivity, guiding further optimization. This work not only establishes a framework for the development of isoform-selective therapeutics but also highlights the potential for unraveling isoform-specific functions in signaling pathways relevant to cancer biology.

ZUSAMMENFASSUNG Proteinkinasen sind wichtige pharmazeutische Targets, doch die Entwicklung selektiver Modulatoren bleibt eine Herausforderung. Auf der Suche nach allosterischen Stellen in der Serin/Threonin‐Kinase p38α existiert eine „Lipidtasche” im C‐Lappen, die ein Potenzial für Bindung kleiner Moleküle aufweist. Die pharmakologische Signifikanz der ursprünglich identifizierten Liganden mit geringer Affinität ist jedoch nicht offensichtlich und wirft die übergeordnete Frage auf, ob eine Art Kommunikation zwischen dieser Tasche und den funktionellen Stellen des Enzyms existiert. Hier verwenden wir NMR‐Spektroskopie, um eine effektive Konnektivität dieser Stellen trotz ihrer räumlichen Distanz aufzuzeigen. Die Daten zeigen eine klare Interdependenz der Proteindynamik zwischen den verschiedenen Strukturelementen durch dynamische Allosterie, was die generelle Möglichkeit der pharmakologischen Entwicklung geeigneter Lipidtaschenliganden nahelegt, um die enzymatische Funktionalität im Krankheitskontext allosterisch zu adressieren.

BACKGROUND:Severe imatinib toxicity is a rare, but relevant event in GIST patients. Other approved treatments were not evaluated within a first-line setting and exhibit severe adverse events that limit QoL. Nilotinib was evaluated within this setting in GIST with a safety profile comparable to imatinib and effectiveness within KIT-exon-11-mutant GIST subgroup (ENESTg1). MATERIALS:, Patients and Methods: This is a retrospective case series reporting the outcome and side effects of 20 patients treated with nilotinib following severe imatinib toxicity. Nilotinib efficacy was tested in GIST cell lines carrying common primary and resistance mutations in KIT. Inhibitory profile of nilotinib was characterized using in silico modeling. RESULTS:In our retrospective analysis of 1263 patients, 7.3% of patients discontinued imatinib due to toxicity. In 20 patients who received nilotinib, reasons were skin (n = 11) and liver (n = 4) toxicity, followed by cardiac (n = 2) and gastrointestinal toxicities (n = 2). No cross-toxicities were observed with nilotinib treatment. Nilotinib was effective after imatinib intolerance. A secondary KIT Exon 9 mutation was found in a progressing lesion. In vitro viability assays showed effectiveness in KIT-Exon-11-mutant cell lines, but not in Exon 9 or imatinib-resistant mutations in doses deemed clinically achievable. In silico modeling revealed steric hindrance by Exon 9 or imatinib resistance mutations. CONCLUSION:Nilotinib exhibits no cross toxicity with imatinib and represents an important alternative in imatinib-sensitive, KIT-exon-11-mutant GIST given its excellent toxicity profile. There is no biological rationale for the use of nilotinib in imatinib-resistant GIST with secondary KIT mutations.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Current tyrosine kinase inhibitors (TKIs) targeting oncogenic KIT and PDGFRA have improved patient outcomes, yet off-target toxicities and drug resistance mutations remain major clinical challenges. Many approved TKIs, often repurposed from other cancer indications, harbor diverse hinge-binding motifs that limit activity against resistance mutations clustering in the ATP-binding pocket of the kinase domain. Here, we describe a structure-based scaffold-hopping strategy to design kinase inhibitors with selectivity for mutant KIT/PDGFRA. Using structure-activity relationship (SAR) studies and 14 determined co-crystal structures, including a structure of the PDGFRA-G680R solvent-front mutation, we define key molecular interactions underlying resistance and inhibitor selectivity. Our lead 6,7-quinazoline-based inhibitors show high potency against clinically relevant KIT/PDGFRA mutations and effectively suppress downstream signaling. These compounds provide selective chemical tools to interrogate resistance mechanisms, and the PDGFRA-G680R structure shows the molecular basis for targeting solvent-front mutations across oncogenic kinases.

Ewing Sarcoma (EwS) is a highly aggressive malignancy predominantly affecting young individuals, with poor prognosis in metastatic or relapsed cases. This study investigates the therapeutic potential of ataxia telangiectasia and Rad3-related protein (ATR) inhibition using the selective small-molecule inhibitor elimusertib, both as a monotherapy and in combination with cytotoxic drugs or radiotherapy. Elimusertib significantly inhibited cell proliferation and induced apoptosis in EwS cell lines. In a chorioallantoic membrane (CAM) model, elimusertib suppressed tumor initiation and reduced tumor volume. Notably, elimusertib exhibited synergistic effects with standard chemotherapeutics and radiation, enhancing antitumor efficacy. These preclinical findings suggest that ATR inhibition by elimusertib may enhance current EwS therapies and enable dose reduction of cytotoxic drugs. Further clinical evaluation is warranted to validate these findings and explore elimusertib’s translational potential in EwS treatment.

ZUSAMMENFASSUNG Die Akt‐Familie der Serin/Threonin‐Kinasen spielt eine entscheidende Rolle bei zellulären Prozessen wie Proliferation, Zellüberleben und Metabolismus. Die drei Akt‐Isoformen (Akt1, Akt2 und Akt3) erfüllen dabei unterschiedliche physiologische Funktionen. Während die Dysregulation einzelner Isoformen mit Erkrankungen wie Krebs assoziiert ist, geht der klinische Einsatz unselektiver Akt‐Inhibitoren häufig mit erheblichen Nebenwirkungen einher. Geleitet von Röntgenkristallstrukturanalysen und Molekülmodellierung wurden strukturelle Merkmale der allosterischen Bindetaschen identifiziert, die die Isoformselektivität bestimmen, und gezielt für das strukturbasierte Design neuer Inhibitoren genutzt. Nach biologischer Charakterisierung erwiesen sich Inhibitoren mit einem Pyrazol‐Grundgerüst als die wirksamsten und selektivsten Akt2‐Inhibitoren, während Inhibitoren mit Pyridin‐Grundgerüst eine ausgeprägte Präferenz für Akt3 zeigten. Darüber hinaus wurden diese neuen Inhibitoren in patientenabgeleiteten kolorektalen Karzinomorganoiden evaluiert. Kokristallstrukturen boten Einblicke in die molekularen Grundlagen der Isoformselektivität und stimulierten das weitere Inhibitordesign. Diese Arbeit liefert damit einen Rahmen für die rationale Entwicklung isoformselektiver Wirkstoffe und unterstreicht deren Potenzial als chemische Sonden zur Aufklärung isoformspezifischer Signalfunktionen in komplexen biologischen Systemen.

Gastrointestinal stromal tumors (GISTs) are primarily driven by activating mutations in the receptor tyrosine kinases KIT or PDGFRA. Targeted therapies have significantly improved patient outcomes; however, acquired drug resistance continues to limit long-term efficacy. Secondary and tertiary mutations across distinct regions of the kinase domain affect the conformational equilibrium or directly disrupt drug binding by alteration of critical residues. This perspective highlights key structural insights that elucidate the molecular basis of resistance and its interplay with kinase activation and TKI binding. A comprehensive understanding of these molecular alterations is crucial for guiding future therapeutic strategies to overcome resistance.

Abstract The protein kinase Akt and its isoforms play a crucial role in various diseases. Unique functions of the individual isoforms (Akt1, Akt2, Akt3) might be essential for survival in malignancies. Particularly for Akt2, it was reported that a knock-out led to diabetic phenotype and might be correlated with clinically adverse hyperglycemic effects observed in pan Akt-treatment. Enduring failure of Akt inhibitors in the clinic indicates the necessity for a thorough understanding of the underlying biology, preferably by using highly isoform-selective small molecules. Here we report the structure-guided development of Akt2-selective covalent-allosteric probe molecules, that can be successfully modified within a complex environment using biorthogonal chemistry. Thus, enabling first Akt2-specific pull-down studies and the use in functional studies, such as selective fluorescent labeling in cellular systems. These chemical probes expand our toolbox to dissect the critical questions of Akt2’s function in health and disease, thereby paving the way for novel therapeutic strategies based on thorough mechanistic insights.

Protein kinases represent major pharmaceutical targets, but the development of selective modulators remains challenging. In search of allosteric sites in the serine/threonine kinase p38α, a "lipid pocket" in the C-lobe has been found to bear prospects for the binding of small molecules. A pharmacological potential of those low-affinity binders found initially has not become obvious, however, raising the overarching question whether any sort of communication between this pocket and the enzyme's functional sites exists. Here, we use NMR spectroscopy to reveal an effective connectivity of these sites in spite of their spatial distance. The data reveal a clear interdependency of protein dynamics between the different structural elements through dynamic allostery, together suggesting a pharmacological avenue for the development of suitable lipid pocket binders to allosterically alter enzymatic functionality in a disease context.

Chemical probes that form a covalent bond with their target protein have been established as a powerful tool for investigating proteins and modulating their activity, but until recently were mostly targeting cysteine residues. Covalent binders that target lysine residues are increasingly reported. Covalent binding to lysine involves challenges such as the increased pKa of the side chain and its considerable flexibility. Here, we describe two computational methods to derivatize lysine-binding covalent small-molecules based on known noncovalent binders, approaching the design problem from two opposite directions. In a "ligand-side" approach, we scan different ligand positions to install an electrophile and dock these derivatized ligands into the target protein. In a "protein-side" approach, we install an electrophile on the target lysine and model its conformational space to find suitable installation vectors on the ligand. We applied both of these protocols retrospectively to a data set of electrophilic ligands and to a data set of vitamin B6 covalently bound to a receptor lysine residue. Our ligand-side protocol successfully identified the known covalent binder in 80% and 86% of cases, while the protein-side protocol achieved identification rates of 56% and 82%, respectively. We prospectively validated these protocols by designing and testing a new lysine-targeting MKK7 inhibitor. Mass-spectrometry and crystallography validated the covalent binding to the target lysine. Applying these protocols to a data set of known kinase inhibitors identified high-confidence covalent candidates for more than 200 human kinases, demonstrating the potential impact of our protocols.

Representativeness of Study participants

PURPOSE:Imatinib resistance is conferred by secondary mutations within the ATP-binding pocket or the activation loop of KIT in gastrointestinal stromal tumors (GIST). Ponatinib is active against KIT secondary activation loop and the gatekeeper mutation T670I in vitro. We evaluated the safety, activity, and inhibitory profile of lower-dose (30 mg) ponatinib in pretreated patients with KIT-mutant GIST. PATIENTS AND METHODS:POETIG was a multicenter phase II trial in patients with advanced, unresectable GIST progressing after imatinib (cohort A, KITV654A-, and cohort B, KITV654A+) or at least imatinib, sunitinib, and regorafenib (cohort C). The primary endpoint was the clinical benefit rate at 16 weeks (modified RECIST 1.1). ctDNA was analyzed for mutations V654A and T670I, and ponatinib was characterized in silico and in vitro using a panel of GIST cell lines. RESULTS:A total of 46 patients were enrolled, and the median follow-up was 1.3 years. The 16-week clinical benefit rate in cohorts A + B was five of 19 (26.3%) and nine of 27 (33.3%) in cohort C, with a median progression-free survival of 2.1 and 3.4 months with four (cohorts A + B) and five (cohort C) patients responding for >6 months. These patients were characterized by exon 11 and/or T670I mutations. Cardiovascular events occurred in four of 46 patients; most common grade 3/4 side effects were hypertension, gamma-glutamyl transferase (GGT)/lipase increase, abdominal pain, and infections. Clinical observations were reproduced by cell line screening and structural modeling. CONCLUSIONS:The trial did not meet its primary endpoint, but lower-dosed ponatinib provided clinical benefit in a subset of patients with expected toxicities. Patients harboring primary exon 11 and/or resistance mutations in T670I and/or exons 17/18 may represent a relevant therapeutic niche for ponatinib.

Efficacy Assessment: CBR, best response

Gastrointestinal stromal tumors (GIST), driven by KIT and PDGFRA mutations, are the most common mesenchymal tumors of the gastrointestinal tract. Although tyrosine kinase inhibitors (TKIs) have advanced treatment, resistance mutations and off-target toxicity limit their efficacy. This study develops covalent TKIs targeting drug-resistant GIST through structure-based design, synthesis, and biological evaluation. SAR studies provided key insights into mutant KIT and PDGFRA interactions, and the first crystal structure of PDGFRA bound to a covalent inhibitor is reported. These findings highlight the promise of covalent inhibitors for overcoming resistance and advancing safer, more effective therapies for advanced GIST.

Ten-eleven translocation (TET) methylcytosine dioxygenases are part of the epigenetic regulatory machinery that erases DNA methylation. Aberrant TET activities are frequently found in hematopoietic malignancies, where loss of TET2 function leads to DNA hypermethylation. A comprehensive understanding of the biological role of TETs is essential to elucidate disease pathogenesis and identify novel therapeutic strategies. We present a robust pipeline integrating protein X-ray crystallography, molecular modeling, and pharmacophore analysis to advance the current TET inhibitor development. In addition, we have synthesized and evaluated a series of 8-hydroxyquinoline (8-HQ) derivatives, demonstrating their potential as chemical tools to explore TET function further. These findings lay the groundwork for a TET-centered therapeutic approach.

Structural Analyses of sunitinib, regorafenib and ripretinib in T670I-mutant KIT

The small GTPase KRAS is a key driver of carcinogenesis when mutated, and significant progress has been made in targeting KRASG12C and other oncogenic variants. Building on our previous work demonstrating the potential of nucleotide-based inhibitors with an acrylamide warhead to target KRASG13C, we designed and synthesized a library of nucleotide-based compounds with cyclic linkers to explore the effect of warhead orientation on reactivity toward Cys13. Using mass spectrometry, kinetic studies, and protein X-ray crystallography, we validated the binding and reactivity of these modulators. In addition, computational predictions of the conformational space of the linkers and warheads provided insights into their reactivity, which agreed well with the experimental data. These findings advance our understanding of the structure-reactivity relationship in these nucleotide-based KRAS inhibitors and will be the basis for further optimization.

Akt kinase is vital in cell growth, survival, metabolism, and migration. Dysregulation of Akt signaling is implicated in cancer and metabolic disorders. In the context of cancer, overactive Akt promotes cell survival and proliferation. This has spurred extensive research into developing Akt inhibitors as potential therapeutic agents to disrupt aberrant Akt signaling. Akt inhibitors are classified into three main types: ATP-competitive, allosteric, and covalent-allosteric inhibitors (CAAIs). ATP-competitive inhibitors compete with ATP for binding to Akt, allosteric inhibitors interact with the Pleckstrin homology (PH) domain, and covalent-allosteric inhibitors form covalent bonds, making them more potent and selective. Notably, capivasertib (AZD5363), a potent ATP-competitive Akt inhibitor, received FDA approval in November 2023 for use in combination with the estrogen receptor degrader fulvestrant to treat breast cancer. Challenges remain, including improving selectivity, identifying biomarkers to tailor treatments, and enhancing therapeutic efficacy while minimizing adverse effects. Particularly covalent-allosteric inhibitors hold promise for future more effective and personalized treatments.