Recent Discovery and Development of Inhibitors that Target CDK9 and Their Therapeutic Indications.

CDK9 is a cyclin-dependent kinase that plays pivotal roles in multiple cellular functions including gene transcription, cell cycle regulation, DNA damage repair, and cellular differentiation. Targeting CDK9 is considered an attractive strategy for antitumor therapy, especially for leukemia and lymphoma. Several potent small molecule inhibitors, exemplified by TG02 (4), have progressed to clinical trials. However, many of them face challenges such as low clinical efficacy and multiple adverse reactions and may necessitate the exploration of novel strategies to lead to success in the clinic. In this perspective, we present a comprehensive overview of the structural characteristics, biological functions, and preclinical status of CDK9 inhibitors. Our focus extends to various types of inhibitors, including pan-inhibitors, selective inhibitors, dual-target inhibitors, degraders, PPI inhibitors, and natural products. The discussion encompasses chemical structures, structure-activity relationships (SARs), biological activities, selectivity, and therapeutic potential, providing detailed insight into the diverse landscape of CDK9 inhibitors.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on SNAr Electrophiles.

Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.

Design, synthesis, biological assessment and molecular modeling studies of novel imidazothiazole-thiazolidinone hybrids as potential anticancer and anti-inflammatory agents.

A new series of imidazothiazole derivatives bearing thiazolidinone moiety (4a-g and 5a-d) were designed, synthesized and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer and anti-inflammatory activity, cardiomyopathy toxicity and hepatotoxicity. Compound 4c inhibited EGFR kinase at a concentration of 18.35 ± 1.25 µM, whereas standard drug erlotinib showed IC50 value of 06.12 ± 0.92 µM. The molecular docking, dynamics simulation and MM-GBSA binding energy calculations revealed strong interaction of compound 4c with binding site of EGFR. The synthesized compounds were evaluated for their anticancer activity by MTT assay against three human cancer cell lines A549 (Lung), MCF-7 (Breast), HCT116 (Colon), one normal human embryonic kidney cell line HEK293 and also for their EGFR kinase inhibitory activity. Few compounds of the series (4a, 4b, 4c) showed promising growth inhibition against all the tested cancer cell lines and against EGFR kinase. Among these, compound 4c was found to be most active and displayed IC50 value of 10.74 ± 0.40, 18.73 ± 0.88 against cancer cell lines A549 and MCF7 respectively whereas it showed an IC50 value of 96.38 ± 1.79 against HEK293 cell line indicating lesser cytotoxicity for healthy cell. Compounds 4a, 4b and 4c were also examined for their apoptosis inducing potential through AO/EB dual staining assay and it was observed that their antiproliferative activity against A549 cells is mediated via induction of apoptosis. Cardiomyopathy studies showed normal cardiomyocytes with no marked sign of pyknotic nucleus of compounds 4b and 4c. Hepatotoxicity studies of compounds 4b and 4c also showed normal architecture of hepatocytes. Compounds 4a-g and 5a-d were also evaluated for their in-vitro anti-inflammatory activity by protein albumin denaturation assay. Among the tested compounds 4a-d and 5a-b showed promising activity and were selected for in-vivo inflammatory activity against carrageenan rat paw edema test. Among these compounds, 4b was found to be most active in the series showing 84.94% inhibition, whereas the standard drug diclofenac sodium showed 84.57% inhibition. Compound 4b also showed low ulcerogenic potential and lipid peroxidation. Thus, compounds 4c and 4b could be a promising lead compounds for developing anticancer and anti-inflammatory agents with low toxicity and selectivity.

Discovery of Potent and Selective PI3Kδ Inhibitors for the Treatment of Acute Myeloid Leukemia.

PI3Kδ is an essential target correlated to the occurrence and development of acute myeloid leukemia (AML). Herein, we investigated the pyrazolo[3,4-d]pyrimidine derivatives as potent and selective PI3Kδ inhibitors with high therapeutic efficacy toward AML. There were 44 compounds designed and prepared in a four-round optimization, and the biological evaluation showed that (S)-36 exhibited potent PI3Kδ inhibitory activity, high selectivity, and high antiproliferative activities against MV-4-11 and MOLM-13 cells, coupled with high oral bioavailability (F = 59.6%). In the MOLM-13 subcutaneous xenograft model, (S)-36 could significantly suppress the tumor progression with a TGI of 67.81% at an oral administration dosage of 10 mg/kg without exhibiting obvious toxicity. Mechanistically, (S)-36 could robustly inhibit the PI3K/AKT pathway for significant suppression of cell proliferation and remarkable induction of apoptosis both in vitro and in vivo. Thus, compound (S)-36 represents a promising PI3Kδ inhibitor for the treatment of acute myeloid leukemia with high efficacy.

Design and Synthesis of Novel Macrocyclic Derivatives as Potent and Selective Cyclin-Dependent Kinase 7 Inhibitors.

The duality of function (cell cycle regulation and gene transcription) of cyclin-dependent kinase 7 (CDK7) makes it an attractive oncology target and the discovery of CDK7 inhibitors has been a long-term pursuit by academia and pharmaceutical companies. However, achieving selective leading compounds is still difficult owing to the similarities among the ATP binding pocket. Herein, we detail the design and synthesis of a series of macrocyclic derivatives with pyrazolo[1,5-a]-1,3,5-triazine core structure as potent and selective CDK7 inhibitors. The diverse manners of macrocyclization led to distinguished selectivity profiles of the CDK family. Molecular dynamics (MD) simulation explained the binding difference between 15- and 16-membered macrocyclic compounds. Further optimization generated compound 37 exhibiting good CDK7 inhibitory activity and high selectivity over other CDKs. This work clearly demonstrated macrocyclization is a versatile method to finely tune the selectivity profile of small molecules and MD simulation can be a valuable tool in prioritizing designs of the macrocycle.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

Akt Inhibitor Advancements: From Capivasertib Approval to Covalent-Allosteric Promises.

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.

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Discovery of novel FGFR4 inhibitors through a build-up fragment strategy.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. FGFR4 has been implicated in HCC progression, making it a promising therapeutic target. We introduce an approach for identifying novel FGFR4 inhibitors by sequentially adding fragments to a common warhead unit. This strategy resulted in the discovery of a potent inhibitor, 4c, with an IC50 of 33 nM and high selectivity among members of the FGFR family. Although further optimisation is required, our approach demonstrated the potential for discovering potent FGFR4 inhibitors for HCC treatment, and provides a useful method for obtaining hit compounds from small fragments.

Targeting EGFR and VEGFR-2 Kinases With Nanoparticles: A Computational Approach for Cancer Therapy Advancement.

The study investigates titanium and zinc nanoparticles as inhibitors for the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), pivotal regulators of cell processes. VEGFR-2 activation fuels tumor angiogenesis in cancer cells, sustaining malignant tissue expansion. Molecular docking analysis illustrates the nanoparticles' binding to the active sites, inhibiting the phosphorylation of key proteins in downstream signaling. This inhibition offers a promising therapeutic approach to impede cancer-related signaling, potentially slowing down aberrant protein cascades controlled by EGFR and VEGFR-2. The findings propose a novel avenue for cancer treatment, targeting abnormal growth pathways using titanium and zinc nanoparticles.

Comprehensive Data-Driven Assessment of Non-Kinase Targets of Inhibitors of the Human Kinome.

Protein kinases (PKs) are involved in many intracellular signal transduction pathways through phosphorylation cascades and have become intensely investigated pharmaceutical targets over the past two decades. Inhibition of PKs using small-molecular inhibitors is a premier strategy for the treatment of diseases in different therapeutic areas that are caused by uncontrolled PK-mediated phosphorylation and aberrant signaling. Most PK inhibitors (PKIs) are directed against the ATP cofactor binding site that is largely conserved across the human kinome comprising 518 wild-type PKs (and many mutant forms). Hence, these PKIs often have varying degrees of multi-PK activity (promiscuity) that is also influenced by factors such as single-site mutations in the cofactor binding region, compound binding kinetics, and residence times. The promiscuity of PKIs is often-but not always-critically important for therapeutic efficacy through polypharmacology. Various in vitro and in vivo studies have also indicated that PKIs have the potential of interacting with additional targets other than PKs, and different secondary cellular targets of individual PKIs have been identified on a case-by-case basis. Given the strong interest in PKs as drug targets, a wealth of PKIs from medicinal chemistry and their activity data from many assays and biological screens have become publicly available over the years. On the basis of these data, for the first time, we conducted a systematic search for non-PK targets of PKIs across the human kinome. Starting from a pool of more than 155,000 curated human PKIs, our large-scale analysis confirmed secondary targets from diverse protein classes for 447 PKIs on the basis of high-confidence activity data. These PKIs were active against 390 human PKs, covering all kinase groups of the kinome and 210 non-PK targets, which included other popular pharmaceutical targets as well as currently unclassified proteins. The target distribution and promiscuity of the 447 PKIs were determined, and different interaction profiles with PK and non-PK targets were identified. As a part of our study, the collection of PKIs with activity against non-PK targets and the associated information are made freely available.

Design, Synthesis, Biological Evaluation and Molecular Docking of Novel F-18-Labeled Focal Adhesion Kinase Inhibitors as Potential Tumor Radiotracers.

Tumor diagnosis, especially at the early stages, holds immense significance. Focal adhesion kinase (FAK) is often highly expressed across various types of tumors, making it a promising target for both therapy and diagnosis. In this study, seven novel inhibitors were designed and synthesized. The inhibitory activity of these compounds against FAK was notably potent, with an IC50 range of 1.27-1968 nM. In particular, compounds 7a and 7c, with IC50 values of 5.59 nM and 1.27 nM, respectively, were radiolabeled with F-18 and then evaluated with S-180 tumor-bearing mice. Subsequently, they exhibited moderate-to-high tumor uptake values, with [18F]7a showing 1.39 ± 0.30%ID/g at 60 min post injection and [18F]7c demonstrating 6.58 ± 0.46%ID/g at 30 min post injection. In addition, the results from docking studies revealed the binding specifics of the studied compounds. Overall, these findings hold the potential to offer valuable guidance for enhancing the development of radiotracers and enzyme inhibitors.

Structural Insights on the Role of Halogen Bonding in Protein MEK Kinase-Inhibitor Complexes.

Kinases are enzymes that play a critical role in governing essential biological processes. Due to their pivotal involvement in cancer cell signaling, they have become key targets in the development of anti-cancer drugs. Among these drugs, those containing the 2,4-dihalophenyl moiety demonstrated significant potential. Here we show how this moiety, particularly the 2-fluoro-4-iodophenyl one, is crucial for the structural stability of the formed drug-enzyme complexes. Crystallographic analysis of reported kinase-inhibitor complex structures highlights the role of the halogen bonding that this moiety forms with specific residues of the kinase binding site. This interaction is not limited to FDA-approved MEK inhibitors, but it is also relevant for other kinase inhibitors, indicating its broad relevance in the design of this class of drugs.

Discovery of macrocyclic CDK2/4/6 inhibitors with improved potency and DMPK properties through a highly efficient macrocyclic drug design platform.

Cyclin-dependent kinases (CDKs) are critical cell cycle regulators that are often overexpressed in tumors, making them promising targets for anti-cancer therapies. Despite substantial advancements in optimizing the selectivity and drug-like properties of CDK inhibitors, safety of multi-target inhibitors remains a significant challenge. Macrocyclization is a promising drug discovery strategy to improve the pharmacological properties of existing compounds. Here we report the development of a macrocyclization platform that enabled the highly efficient discovery of a novel, macrocyclic CDK2/4/6 inhibitor from an acyclic precursor (NUV422). Using dihedral angle scan and structure-based, computer-aided drug design to select an optimal ring-closing site and linker length for the macrocycle, we identified compound 8 as a potent new CDK2/4/6 inhibitor with optimized cellular potency and safety profile compared to NUV422. Our platform leverages both experimentally-solved as well as generative chemistry-derived macrocyclic structures and can be deployed to streamline the design of macrocyclic new drugs from acyclic starting compounds, yielding macrocyclic compounds with enhanced potency and improved drug-like properties.

Chemical Scaffolds for the Clinical Development of Mutant-Selective and Reversible Fourth-Generation EGFR-TKIs in NSCLC.

In nonsmall cell lung cancer (NSCLC), as well as in other tumors, the targeted therapy is mainly represented by tyrosine kinase inhibitors (TKIs), small molecules able to target oncogenic driver alterations affecting the gene encoding the epidermal growth factor receptor (EGFR). Up to now, several different TKIs have been developed. However, cancer cells showed an incredible adaptive tumor response to the inhibition of the sequentially mutated EGFR (EGFRM+), triggering the need to explore novel pharmacochemical strategies. This Review summarizes the recent efforts in the development of new reversible next-generation EGFR TKIs to fight the resistance against T790M and C797S mutations. Specifically, after giving an overview of the role of the EGFR's signaling pathways in cancer progression, we are going to discuss the most relevant approved drugs and drug candidates in terms of chemical structure, binding modalities, and their potency and selectivity against the mutated EGFR over the wild-type form. This could provide important guidelines and rationale for the discovery and iterative development of new drugs.

Recent development of VEGFR small molecule inhibitors as anticancer agents: A patent review (2021-2023).

VEGFR, a receptor tyrosine kinase inhibitor (TKI), is an important regulatory factor that promotes angiogenesis and vascular permeability. It plays a significant role in processes such as tumor angiogenesis, tumor cell invasion, and metastasis. VEGFR is mainly composed of three subtypes: VEGFR-1, VEGFR-2, and VEGFR-3. Among them, VEGFR-2 is the crucial signaling receptor for VEGF, which is involved in various pathological and physiological functions. At present, VEGFR-2 is closely related to a variety of cancers, such as non-small cell lung cancer (NSCLC), Hepatocellular carcinoma, Renal cell carcinoma, breast cancer, gastric cancer, glioma, etc. Consequently, VEGFR-2 serves as a crucial target for various cancer treatments. An increasing number of VEGFR inhibitors have been discovered to treat cancer, and they have achieved tremendous success in the clinic. Nevertheless, VEGFR inhibitors often exhibit severe cytotoxicity, resistance, and limitations in indications, which weaken the clinical therapeutic effect. In recent years, many small molecule inhibitors targeting VEGFR have been identified with anti-drug resistance, lower cytotoxicity, and better affinity. Here, we provide an overview of the structure and physiological functions of VEGFR, as well as some VEGFR inhibitors currently in clinical use. Also, we summarize the in vivo and in vitro activities, selectivity, structure-activity relationship, and therapeutic or preventive use of VEGFR small molecule inhibitors reported in patents in the past three years (2021-2023), thereby presenting the prospects and insights for the future development of targeted VEGFR inhibitors.

Imidazo[1,2-b]pyridazines as inhibitors of DYRK kinases.

Selective inhibitors of DYRK1A are of interest for the treatment of cancer, Type 2 diabetes and neurological disorders. Optimization of imidazo [1,2-b]pyridazine fragment 1 through structure-activity relationship exploration and in silico drug design efforts led to the discovery of compound 17 as a potent cellular inhibitor of DYRK1A with selectivity over much of the kinome. The binding mode of compound 17 was elucidated with X-ray crystallography, facilitating the rational design of compound 29, an imidazo [1,2-b]pyridazine with improved kinase selectivity with respect to closely related CLK kinases.

Synthesis, kinase inhibition and anti-leukemic activities of diversely substituted indolopyrazolocarbazoles.

Pyrazole analogues of the staurosporine aglycone K252c, in which the lactam ring was replaced by a pyrazole moiety, were synthesized. In this series, one or the other nitrogen atoms of the indolocarbazole scaffold was substituted by aminoalkyl chains, aiming at improving protein kinase inhibition as well as cellular potency toward acute myeloid leukemia (AML) cell lines. Compound 19a, substituted at the N12-position by a 3-(methylamino)propyl group, showed high cellular activity in the low micromolar range toward three AML cell lines (MOLM-13, OCI-AML3 and MV4-11) with selectivity over non-cancerous cells (NRK, H9c2). 19a is also a highly potent inhibitor of the three Pim kinase isoforms, Pim-3 being the most inhibited with an IC50 value in the nanomolar range. A selectivity screening toward a panel of 50 protein kinases showed that 19a also potently inhibited PRK2 and to a lower extent AMPK, MARK3, GSK3ß and JAK3. Our results enhance the understanding of the structural characteristics of indolopyrazolocarbazoles essential for potent protein kinase inhibition with therapeutic potential against AML.

Classifying protein kinase conformations with machine learning.

Protein kinases are key actors of signaling networks and important drug targets. They cycle between active and inactive conformations, distinguished by a few elements within the catalytic domain. One is the activation loop, whose conserved DFG motif can occupy DFG-in, DFG-out, and some rarer conformations. Annotation and classification of the structural kinome are important, as different conformations can be targeted by different inhibitors and activators. Valuable resources exist; however, large-scale applications will benefit from increased automation and interpretability of structural annotation. Interpretable machine learning models are described for this purpose, based on ensembles of decision trees. To train them, a set of catalytic domain sequences and structures was collected, somewhat larger and more diverse than existing resources. The structures were clustered based on the DFG conformation and manually annotated. They were then used as training input. Two main models were constructed, which distinguished active/inactive and in/out/other DFG conformations. They considered initially 1692 structural variables, spanning the whole catalytic domain, then identified ("learned") a small subset that sufficed for accurate classification. The first model correctly labeled all but 3 of 3289 structures as active or inactive, while the second assigned the correct DFG label to all but 17 of 8826 structures. The most potent classifying variables were all related to well-known structural elements in or near the activation loop and their ranking gives insights into the conformational preferences. The models were used to automatically annotate 3850 kinase structures predicted recently with the Alphafold2 tool, showing that Alphafold2 reproduced the active/inactive but not the DFG-in proportions seen in the Protein Data Bank. We expect the models will be useful for understanding and engineering kinases.