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.

The molecular interaction pattern of lenvatinib enables inhibition of wild-type or kinase-mutated FGFR2-driven cholangiocarcinoma.

Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers.

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

A patent review of MAPK inhibitors (2018 - present).

INTRODUCTION: The mitogen-activated protein kinase (MAPK) family consist of p38 MAP kinases, c-Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs). They are involved in a multitude of diseases, including inflammatory, autoimmune, neurodegenerative, and metabolic diseases as well as cancer. In recent years, further developments in the field of MAPK-inhibitors have been reported, including an isoform or downstream target selective inhibition of MAPKs as well as target protein degradation approaches. AREAS COVERED: This review summarizes newly patented MAPK-inhibitors that were claimed between 2018 and early 2023. Presented are the patents as well as their corresponding publications, the storyline of development, and clinical trials involving these compounds. This article elaborates a total of 27 patents, which were identified using established search engines. EXPERT OPINION: Although industrial research on MAPK-inhibitors has been ongoing for more than 20 years, novel clinical trials of MAPK-inhibitors as potential drug candidates are still being conducted in the period under review. Recently reported inhibitors show an excellent selectivity profile and are even achieving selectivity between closely related isoforms. This progression offers the possibility to eliminate unwanted side effects and may finally lead to the approval of the first MAPK-inhibitor.

Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

Targeting SARS-CoV-2 Main Protease (MPro) with Kinase Inhibitors: A Promising Approach for Discovering Antiviral and Anti-inflammatory Molecules against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infected over 688 million people worldwide, causing public health concern and approximately 6.8 million deaths due to COVID-19. COVID-19, especially severe cases, is characterized by exacerbated lung inflammation with an increase of pro-inflammatory cytokines. In addition to antiviral drugs, there is a need for anti-inflammatory therapies to treat all phases of COVID-19. One of the most attractive drug targets for COVID-19 is the SARS-CoV-2 main protease (MPro), an enzyme responsible for cleaving polyproteins formed after the translation of viral RNA, which is essential for viral replication. MPro inhibitors, therefore, have the potential to stop viral replication and act as antiviral drugs. Considering that several kinase inhibitors are known for their action in inflammatory pathways, this could also be investigated toward a potential anti-inflammatory treatment for COVID-19. Therefore, the use of kinase inhibitors against SARS-CoV-2 MPro may be a promising strategy to find molecules with dual activity─antiviral and anti-inflammatory. Considering this, the potential of six kinase inhibitors against SARS-CoV-2 MPro were evaluated in silico and in vitro, including Baricitinib, Tofacitinib, Ruxolitinib, BIRB-796, Skepinone-L, and Sorafenib. To assess the inhibitory potential of the kinase inhibitors, a continuous fluorescent-based enzyme activity assay was optimized with SARS-CoV-2 MPro and MCA-AVLQSGFR-K(Dnp)-K-NH2 (substrate). BIRB-796 and Baricitinib were identified as inhibitors of SARS-CoV-2 MPro, presenting IC50 values of 7.99 and 25.31 µM, respectively. As they are also known for their anti-inflammatory action, both are prototype compounds with the potential to present antiviral and anti-inflammatory activity against SARS-CoV-2 infection.

Selective Inhibitors of Janus Kinase 3 Modify Responses to Lipopolysaccharides by Increasing the Interleukin-10-to-Tumor Necrosis Factor α Ratio.

Janus kinase (JAK) inhibitors act at low doses (e.g., tofacitinib, 0.2-0.4 µmol/kg bid) in clinical use, suggesting an efficient underlying mode of action. We hypothesized that their effectiveness is due to their ability to raise the ratio of IL-10 to TNFα. Unlike other JAK isoforms, JAK3 is expressed mainly in hematopoietic cells and is essential for immune function. We used JAK3 selective inhibitors with preferential distribution to immune cells. Inhibition of JAK3 in human leukocytes reduced TNFα and IL-6 but maintained levels of IL-10, while pan-JAK inhibitors increased TNFα, IL-6, and IL-10. JAK1 is required for IL-10 receptor signaling, which suggests that, at exposure above the IC50 (55 nM for tofacitinib on JAK1), there is less feedback control of TNFα levels. This leads to self-limiting effects of JAK1 inhibitors and could place an upper limit on appropriate doses. In vivo, treating mice with JAK3 inhibitors before LPS administration decreased plasma TNFα and increased IL-10 above vehicle levels, suggesting that JAK3 inhibition may limit TNFα release by increasing IL-10 while leaving the IL-10 receptor functional. This mechanism should have general utility in controlling autoimmune diseases and can be conveniently observed by measuring the ratio of IL-10 to TNFα. In summary, our targeted, "leukotropic" inhibitors more effectively increased IL-10/TNFα ratios than unselective control compounds and could, therefore, be ideal for autoimmune therapy.

Structural elements that enable specificity for mutant EGFR kinase domains with next-generation small-molecule inhibitors.

Specificity for a desired enzyme target is an essential property of small-molecule inhibitors. Molecules targeting oncogenic driver mutations in the epidermal growth factor receptor (EGFR) kinase domain have had a considerable clinical impact due to their selective binding to cancer-causing mutants compared to wild type. Despite the availability of clinically approved drugs for cancers driven by EGFR mutants, persistent challenges in drug resistance in the past decades have led to newer generations of drugs with divergent chemical structures. The current clinical challenges are mainly due to acquired resistance to third-generation inhibitors, including by the acquisition of the C797S mutation. Several diverse fourth-generation candidates and tool compounds that inhibit the C797S mutant have emerged, and their structural characterization has revealed molecular factors that allow for EGFR mutant selective binding. Here, we have reviewed all known structurally-characterized EGFR TKIs targeting clinically-relevant mutations to identify specific features that enable C797S inhibition. Newer generation EGFR inhibitors exhibit consistent and previously underutilized hydrogen bonding interactions with the conserved K745 and D855 residue side chains. We also consider binding modes and hydrogen bonding interactions of inhibitors targeting the classical ATP and the more unique allosteric sites.

Structural Basis for Inhibition of Mutant EGFR with Lazertinib (YH25448).

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure-Activity Relationships, Antiviral Activity, and X-ray Structure Determination.

The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M-1 s-1 (Ki = 0.0141 µM) and 27,200 M-1 s-1 (Ki = 0.0332 µM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.

Gefitinib-Tamoxifen Hybrid Ligands as Potent Agents against Triple-Negative Breast Cancer.

Anticancer drug conjugates may benefit from simultaneous action at two targets potentially overcoming the drawbacks of current cancer treatment, such as insufficient efficacy, high toxicity, and development of resistance. Compared to a combination of two single-target drugs, they may offer an advantage of pharmacokinetic simplicity and fewer drug-drug interactions. Here, we report a series of compounds connecting tamoxifen or endoxifen with the EGFR-inhibitor gefitinib via a covalent linkage. These hybrid ligands retain both ER antagonist activity and EGFR inhibition. The most potent analogues exhibited single-digit nanomolar activities at both targets. The amide-linked endoxifen-gefitinib drug conjugates 17b and 17c demonstrated the most favorable anti-cancer profile in cellular viability assays on MCF7, MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells. Most importantly, in TNBC cells 17b and 17c displayed nanomolar IC50-values (380 nM - 970 nM) and were superior in their anti-cancer activity compared to their control compounds and combinations thereof.

Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors.

Stress-induced p38α mitogen-activated protein (MAP) kinase activation modulates cytokine overproduction and is associated with neuroinflammation and neurodegeneration. As a potential therapeutic approach, novel Skepinone-based p38α MAP kinase inhibitors were optimized to cross the blood-brain barrier via either amino acid transporters or hydrophobic diffusion. To enhance absorption from the oral route, we used methyl ester prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (43; p38α, IC50 = 5.5 nM) and 4-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC50 = 12 nM) had brain-to-plasma ratios of 1.4 and 4.4, respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (p38α, IC50 = 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

Addressing a Trapped High-Energy Water: Design and Synthesis of Highly Potent Pyrimidoindole-Based Glycogen Synthase Kinase-3ß Inhibitors.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC50 value of our co-crystallized glycogen synthase kinase-3ß (GSK-3ß) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

Design of a "Two-in-One" Mutant-Selective Epidermal Growth Factor Receptor Inhibitor That Spans the Orthosteric and Allosteric Sites.

Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 µM) and L858R/T790M (4.4 µM) variants.

Anticancer Drug Conjugates Incorporating Estrogen Receptor Ligands.

Hormone-dependent cancers, such as certain types of breast cancer are characterized by over-expression of estrogen receptors (ERs). Anticancer drug conjugates combining ER ligands with other classes of anticancer agents may not only benefit from dual action at both anti-cancer targets but also from selective delivery of cytotoxic agents to ER-positive tumor cells resulting in less toxicity and adverse effects. Moreover, they could also take advantage of overcoming resistance typical for anti-hormonal monotherapy such as tamoxifen. In this review, we discuss the design, structures and pharmacological effects of numerous series of drug conjugates containing ER ligands such as selective ER modulators (tamoxifen, 4-hydroxytamoxifen, endoxifen), selective ER degraders (ICI-164384) and ER agonists (estradiol) linked to diverse anti-cancer agents including histone-deacetylase inhibitors, DNA-alkylating agents, antimitotic agents and epidermal growth factor receptor inhibitors.

The pre-clinical discovery and development of osimertinib used to treat non-small cell lung cancer.

Introduction: Osimertinib is currently the only FDA- and EMA-approved third-generation small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). It was initially indicated for second-line treatment of patients with metastatic EGFR T790M mutation-positive non-small cell lung cancer (NSCLC) and got approved for first-line treatment of EGFR activation mutation-positive metastatic NSCLC in 2018. Most recently, the FDA granted approval for the adjuvant treatment of patients with early-stage mutated EGFR NSCLC after tumor resection.Areas covered: This drug discovery case history focuses on the key studies that led to the preclinical discovery and development of osimertinib. The authors focus on published preclinical studies by scientists from AstraZeneca and highlight key events in the clinical development.Expert opinion: Although eventually compromised by the cellular plasticity of the tumor and the inevitable acquisition of drug resistance through the use of osimertinib, its key role in the treatment of NSCLC with specific EGFR mutations will be maintained in the near future. As the genome of EGFR is highly labile and since the rapid development of new mutants remains an issue, there is still room for improvement for the next generation of inhibitors.

From off-to on-target: New BRAF-inhibitor-template-derived compounds selectively targeting mitogen activated protein kinase kinase 4 (MKK4).

The mitogen-activated protein kinase (MAP) kinase 4 (MKK4) was found to be a major regulator of liver regeneration and could be a valuable drug target addressing liver related diseases by restoring its intrinsic regenerative capacity. We report on the synthesis and optimization of novel MKK4 inhibitors following a target-hopping strategy from the FDA-approved BRAFV600E inhibitor PLX4032 (8). Applying an iterative multi-parameter optimization process we carved out essential structural features yielding in compounds with a low nanomolar affinity for MKK4 and excellent selectivity profiles against the main off-targets MKK7 and JNK1, which, upon relevant inhibition, would totally abrogate the pro-regenerative effect of MKK4 inhibition, as well as against the off-targets MAP4K5, ZAK and BRAF with selectivity factors ranging from 40 to 430 for our best-balanced compounds 70 and 73.

Structural Basis for EGFR Mutant Inhibition by Trisubstituted Imidazole Inhibitors.

Acquired drug resistance in epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer is a persistent challenge in cancer therapy. Previous studies of trisubstituted imidazole inhibitors led to the serendipitous discovery of inhibitors that target the drug resistant EGFR(L858R/T790M/C797S) mutant with nanomolar potencies in a reversible binding mechanism. To dissect the molecular basis for their activity, we determined the binding modes of several trisubstituted imidazole inhibitors in complex with the EGFR kinase domain with X-ray crystallography. These structures reveal that the imidazole core acts as an H-bond acceptor for the catalytic lysine (K745) in the "αC-helix out" inactive state. Selective N-methylation of the H-bond accepting nitrogen ablates inhibitor potency, confirming the role of the K745 H-bond in potent, noncovalent inhibition of the C797S variant. Insights from these studies offer new strategies for developing next generation inhibitors targeting EGFR in non-small-cell lung cancer.

An updated patent review of p38 MAP kinase inhibitors (2014-2019).

Introduction: During the first half of the last decade the p38 MAP kinase family was a very popular target in academic as well as industrial research programs. Many attempts to achieve marketing authorization for a p38 MAPK inhibitor for the treatment of pro-inflammatory diseases, like rheumatoid arthritis (RA), failed at the state of clinical trials, mostly due to selectivity and/or toxicity issues.Areas covered: Herein, the patents and corresponding publications of international companies, universities and other research institutions, which focus on the development, identification and optimization of new selective p38 inhibitors and their fields of use are summarized.Expert opinion: p38 MAP kinase inhibitors are a mature field with many pre-clinically validated structural classes, more than 20 candidates in clinical trials but still (except the weak and unselective p38 inhibitor pirfenidone) no approved drug. Big Pharma hasn't contributed much to the patents of the last five years but remarkable contribution have come from academic environment or small biotech companies. Three general punchlines of innovation have shown up. Tailor-made molecules with properties for local application, mainly type-II (Urea-type) inhibitors for lung- or skin diseases, isoform p38γ,δ-selective inhibitors for the treatment of cutaneous t-cell lymphoma (CTCL) and substrate-specific inhibitors (e.g. p38/MK2).