Insights into the Kinetics of the Resistance Formation of Bacteria against Ciprofloxacin Poly(2-methyl-2-oxazoline) Conjugates.

The influence on the resistance formation of polymers attached to antibiotics has rarely been investigated. In this study, ciprofloxacin (CIP) was conjugated to poly(2-methyl-2-oxazoline)s with an ethylene diamine end group (Me-PMOx28-EDA) via two different spacers (CIP modified with α,α'-dichloro- p-xylene-xCIP, CIP modified with chloroacetyl chloride-eCIP). The antibacterial activity of the conjugates against a number of bacterial strains shows a great dependence on the nature of the spacer. The Me-PMOx39-EDA-eCIP, containing a potentially cleavable linker, does not exhibit a molecular weight dependence on antibacterial activity in contrast to Me-PMOx27-EDA-xCIP. The resistance formation of both conjugates against Staphylococcus aureus and Escherichia coli was investigated. Both conjugates showed the potential to significantly delay the formation of resistant bacteria compared to the unmodified CIP. Closer inspection of a possible resistance mechanism by genome sequencing of the topoisomerase IV region of resistant S. aureus revealed that this bacterium mutates at the same position when building up resistance to CIP and to Me-PMOx27-EDA-xCIP. However, the S. aureus cells that became resistant against the polymer conjugate are fully susceptible to CIP. Thus, conjugation of CIP with PMOx seems to alter the resistance mechanism.

Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer.

The treatment of non-small cell lung cancer (NSCLC) is currently experiencing a revolution. Over the last decade, the knowledge gained about the biochemical features of biomarkers and their predictive abilities has led to the development of targeted small-molecule inhibitors that present an alternative to harsh chemotherapy. The use of these new therapies has improved the quality of life and increased the survival of patients. The occurrence of inevitable drug resistance requires the constant development of precision medicine. The detailed understanding of the target biology and the search for innovative chemical approaches has encouraged investigations in this field. Herein, we review selected aspects of the molecular targets and present an overview of current topics and challenges in the rational development of small molecules to target NSCLC.

An Unusual Intramolecular Halogen Bond Guides Conformational Selection.

PIK-75 is a phosphoinositide-3-kinase (PI3K) α-isoform-selective inhibitor with high potency. Although published structure-activity relationship data show the importance of the NO2 and the Br substituents in PIK-75, none of the published studies could correctly determine the underlying reason for their importance. In this publication, we report the first X-ray crystal structure of PIK-75 in complex with the kinase GSK-3ß. The structure shows an unusual U-shaped conformation of PIK-75 within the active site of GSK-3ß that is likely stabilized by an atypical intramolecular Br⋅⋅⋅NO2 halogen bond. NMR and MD simulations show that this conformation presumably also exists in solution and leads to a binding-competent preorganization of the PIK-75 molecule, thus explaining its high potency. We therefore suggest that the site-specific incorporation of halogen bonds could be generally used to design conformationally restricted bioactive substances with increased potencies.

Insight into the Inhibition of Drug-Resistant Mutants of the Receptor Tyrosine Kinase EGFR.

Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.

KIT ATP-Binding Pocket/Activation Loop Mutations in GI Stromal Tumor: Emerging Mechanisms of Kinase Inhibitor Escape.

PURPOSE: Imatinib resistance in GI stromal tumors (GISTs) is primarily caused by secondary KIT mutations, and clonal heterogeneity of these secondary mutations represents a major treatment obstacle. KIT inhibitors used after imatinib have clinical activity, albeit with limited benefit. Ripretinib is a potent inhibitor of secondary KIT mutations in the activation loop (AL). However, clinical benefit in fourth line remains limited and the molecular mechanisms of ripretinib resistance are largely unknown. PATIENTS AND METHODS: Progressing lesions of 25 patients with GISTs refractory to ripretinib were sequenced for KIT resistance mutations. Resistant genotypes were validated and characterized using novel cell line models and in silico modeling. RESULTS: GISTs progressing on ripretinib were enriched for secondary mutations in the ATP-binding pocket (AP), which frequently occur in cis with preexisting AL mutations, resulting in highly resistant AP/AL genotypes. AP/AL mutations were rarely observed in a cohort of progressing GIST samples from the preripretinib era but represented 50% of secondary KIT mutations in patients with tumors resistant to ripretinib. In GIST cell lines harboring secondary KIT AL mutations, the sole genomic escape mechanisms during ripretinib drug selection were AP/AL mutations. Ripretinib and sunitinib synergize against mixed clones with secondary AP or AL mutants but do not suppress clones with AP/AL genotypes. CONCLUSION: Our findings underscore that KIT remains the central oncogenic driver even in late lines of GIST therapy. KIT-inhibitor combinations may suppress resistance because of secondary KIT mutations. However, the emergence of KIT AP/AL mutations after ripretinib treatment calls for new strategies in the development of next-generation KIT inhibitors.

Insight into Targeting Exon20 Insertion Mutations of the Epidermal Growth Factor Receptor with Wild Type-Sparing Inhibitors.

Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1H-pyrrolo[2,3-b]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.

Resistance to Avapritinib in PDGFRA-Driven GIST Is Caused by Secondary Mutations in the PDGFRA Kinase Domain.

Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance. SIGNIFICANCE: Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST.This article is highlighted in the In This Issue feature, p. 1.

Structure Defines Function: Clinically Relevant Mutations in ErbB Kinases.

The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provide valuable insights into their molecular functions. This field has undergone a revolution because X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations. Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility.

Complex Crystal Structures of EGFR with Third-Generation Kinase Inhibitors and Simultaneously Bound Allosteric Ligands.

Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) and currently the gold-standard for the treatment of patients suffering from non-small cell lung cancer (NSCLC) harboring T790M-mutated epidermal growth factor receptor (EGFR). The outcome of the treatment, however, is limited by the emergence of the C797S resistance mutation. Allosteric inhibitors have a different mode of action and were developed to overcome this limitation. However, most of these innovative molecules are not effective as a single agent. Recently, mutated EGFR was successfully addressed with osimertinib combined with the allosteric inhibitor JBJ-04-125-02, but surprisingly, structural insights into their binding mode were lacking. Here, we present the first complex crystal structures of mutant EGFR in complex with third-generation inhibitors such as osimertinib and mavelertinib in the presence of simultaneously bound allosteric inhibitors. These structures highlight the possibility of further combinations targeting EGFR and lay the foundation for hybrid inhibitors as next-generation TKIs.

Targeting Her2-insYVMA with Covalent Inhibitors-A Focused Compound Screening and Structure-Based Design Approach.

Mutated or amplified Her2 serves as a driver of non-small cell lung cancer or mediates resistance toward the inhibition of its family member epidermal growth factor receptor with small-molecule inhibitors. To date, small-molecule inhibitors targeting Her2 which can be used in clinical routine are lacking, and therefore, the development of novel inhibitors was undertaken. In this study, the well-established pyrrolopyrimidine scaffold was modified with structural motifs identified from a screening campaign with more than 1600 compounds, which were applied against wild-type Her2 and its mutant variant Her2-A775_G776insYVMA. The resulting inhibitors were designed to covalently target a reactive cysteine in the binding site of Her2 and were further optimized by means of structure-based drug design utilizing a set of obtained complex crystal structures. In addition, the analysis of binding kinetics and absorption, distribution, metabolism, and excretion parameters as well as mass spectrometry experiments and western blot analysis substantiated our approach.

Characterization of Covalent Pyrazolopyrimidine-MKK7 Complexes and a Report on a Unique DFG-in/Leu-in Conformation of Mitogen-Activated Protein Kinase Kinase 7 (MKK7).

Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.

Targeting the MKK7-JNK (Mitogen-Activated Protein Kinase Kinase 7-c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors.

The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.

A novel scaffold for EGFR inhibition: Introducing N-(3-(3-phenylureido)quinoxalin-6-yl) acrylamide derivatives.

Clinical data acquired over the last decade on non-small cell lung cancer (NSCLC) treatment with small molecular weight Epidermal Growth Factor Receptor (EGFR) inhibitors have shown significant influence of EGFR point mutations and in-frame deletions on clinical efficacy. Identification of small molecules capable of inhibiting the clinically relevant EGFR mutant forms is desirable, and novel chemical scaffolds might provide knowledge regarding selectivity among EGFR forms and shed light on new strategies to overcome current clinical limitations. Design, synthesis, docking studies and in vitro evaluation of N-(3-(3-phenylureido)quinoxalin-6-yl) acrylamide derivatives (7a-m) against EGFR mutant forms are described. Compounds 7h and 7l were biochemically active in the nanomolar range against EGFRwt and EGFRL858R. Molecular docking and reaction enthalpy calculations have shown the influence of the combination of reversible and covalent binding modes with EGFR on the inhibitory activity. The inhibitory profile of 7h against a panel of patient-derived tumor cell lines was established, demonstrating selective growth inhibition of EGFR related cells at 10 µM among a panel of 30 cell lines derived from colon, melanoma, breast, bladder, kidney, prostate, pancreas and ovary tumors.

Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S.

Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.

C797S Resistance: The Undruggable EGFR Mutation in Non-Small Cell Lung Cancer?

The first evidence of osimertinib resistance mediated by the epidermal growth factor receptor (EGFR) mutation C797S was reported three years ago. Since then, no major breakthroughs have been achieved to target the clinically relevant mutant variant that impedes covalent bond formation with irreversible EGFR inhibitors. Although several biochemically active compounds have been described, only a few inhibitors that potently act on the cellular level or in vivo have been introduced so far. Herein, we give an overview of current approaches in the field and highlight the challenges that need to be addressed in future research projects to overcome the C797S-mediated drug resistance.

RASPELD to Perform High-End Screening in an Academic Environment toward the Development of Cancer Therapeutics.

The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing.

Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors.

The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors.

Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor.

The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.

Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site.

Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure-activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant.

Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor.

Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.