Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update.

Covalent inhibitors and other types of covalent modalities have seen a revival in the past two decades, with a variety of new targeted covalent drugs having been approved in recent years. A key feature of such molecules is an intrinsically reactive group, typically a weak electrophile, which enables the irreversible or reversible formation of a covalent bond with a specific amino acid of the target protein. This reactive group, often called the "warhead", is a critical determinant of the ligand's activity, selectivity, and general biological properties. In 2019, we summarized emerging and re-emerging warhead chemistries to target cysteine and other amino acids (Gehringer, M.; Laufer, S. A. J. Med. Chem. 2019, 62, 5673-5724; DOI: 10.1021/acs.jmedchem.8b01153). Since then, the field has rapidly evolved. Here we discuss the progress on covalent warheads made since our last Perspective and their application in medicinal chemistry and chemical biology.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Targeted Covalent Inhibitors in Drug Discovery, Chemical Biology and Beyond.

Covalent inhibitors have experienced a revival in medicinal chemistry and chemical biology in recent decades [...].

Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B.

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Chemical Probes for Understudied Kinases: Challenges and Opportunities.

Over 20 years after the approval of the first-in-class protein kinase inhibitor imatinib, the biological function of a significant fraction of the human kinome remains poorly understood while most research continues to be focused on few well-validated targets. Given the strong genetic evidence for involvement of many kinases in health and disease, the understudied fraction of the kinome holds a large and unexplored potential for future therapies. Specific chemical probes are indispensable tools to interrogate biology enabling proper preclinical validation of novel kinase targets. In this Perspective, we highlight recent case studies illustrating the development of high-quality chemical probes for less-studied kinases and their application in target validation. We spotlight emerging techniques and approaches employed in the generation of chemical probes for protein kinases and beyond and discuss the associated challenges and opportunities.

Discovery of a Potent and Highly Isoform-Selective Inhibitor of the Neglected Ribosomal Protein S6 Kinase Beta 2 (S6K2).

The ribosomal protein S6 kinase beta 2 (S6K2) is thought to play an important role in malignant cell proliferation, but is understudied compared to its closely related homolog S6 kinase beta 1 (S6K1). To better understand the biological function of S6K2, chemical probes are needed, but the high similarity between S6K2 and S6K1 makes it challenging to selectively address S6K2 with small molecules. We were able to design the first potent and highly isoform-specific S6K2 inhibitor from a known S6K1-selective inhibitor, which was merged with a covalent inhibitor engaging a cysteine located in the hinge region in the fibroblast growth factor receptor kinase (FGFR) 4 via a nucleophilic aromatic substitution (SNAr) reaction. The title compound shows a high selectivity over kinases with an equivalently positioned cysteine, as well as in a larger kinase panel. A good stability towards glutathione and Nα-acetyl lysine indicates a non-promiscuous reactivity pattern. Thus, the title compound represents an important step towards a high-quality chemical probe to study S6K2-specific signaling.

Discovery of a Potent Dual SLK/STK10 Inhibitor Based on a Maleimide Scaffold.

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

Development of Pyridine-based Inhibitors for the Human Vaccinia-related Kinases 1 and 2.

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (26) displayed an IC50 value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04). Differences in compound binding mode and substituent preferences between the two VRKs were identified by the structure-activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point for the design of more specific and potent inhibitors against each of the two VRKs.

The latest advances in the discovery of nitric oxide hybrid drug compounds.

INTRODUCTION: There is a great interest in Nitric oxide (NO) within medicinal chemistry since it's involved in human signaling pathways. Prodrugs or hybrid compounds containing NO-donor scaffolds linked to an active compound are valuable, due to their potential for modulating many pathological conditions due to NO's biological properties when released in addition to the native drug. Compounds that selectively inhibit nitric oxide synthase isoforms (NOS) can also increase therapeutic capacity, particularly in the treatment of chronic diseases. However, search for bioactive compounds to efficiently and selectively modulate NO is still a challenge in drug discovery. Areas covered: In this review, the authors highlight the recent advances in the strategies used to discover NO-hybrid derivatives, especially those related to anti-inflammatory, cardiovascular, anticancer and anti-microorganism activities. They also focus on: nitric oxide synthase inhibitors, NO delivery materials and other related activities. Expert opinion: The process of molecular hybridization can be used to obtain NO-releasing compounds that also interact with different targets. The main problem with this approach is to control NO multiple actions in the right biological system. However, the use of NO-releasing groups with many different scaffolds leads to new molecular structures for bioactive compounds, suggesting synergies.

New antibacterial agents: Hybrid bioisoster derivatives as potential E. coli FabH inhibitors.

The development of resistance to antibiotics by microorganisms is a major problem for the treatment of bacterial infections worldwide, and therefore, it is imperative to study new scaffolds that are potentially useful in the development of new antibiotics. In this regard, we propose the design, synthesis and biological evaluation of hybrid sulfonylhydrazone bioisosters/furoxans with potential antibacterial (Escherichia coli) activity. The most active compound of the series, (E)-3-methyl-4-((2-tosylhydrazono)methyl)-1,2,5-oxadiazole 2-oxide, with a MIC=0.36µM, was not cytotoxic when tested on Vero cells (IC50>100µM). To complement the in vitro screening, we also studied the interaction of the test compounds with ß-ketoacyl acyl carrier protein synthase (FabH), the target for the parent compounds, and we observed three important hydrogen-bonding interactions with two important active site residues in the catalytic site of the enzyme, providing complementary evidence to support the target of the new hybrid molecules.