Discovery of potent and selective HER2 inhibitors with efficacy against HER2 exon 20 insertion-driven tumors, which preserve wild-type EGFR signaling.

Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.

Selective and Potent CDK8/19 Inhibitors Enhance NK-Cell Activity and Promote Tumor Surveillance.

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.

Start Selective and Rigidify: The Discovery Path toward a Next Generation of EGFR Tyrosine Kinase Inhibitors.

The epidermal growth factor receptor (EGFR), when carrying an activating mutation like del19 or L858R, acts as an oncogenic driver in a subset of lung tumors. While tumor responses to tyrosine kinase inhibitors (TKIs) are accompanied by marked tumor shrinkage, the response is usually not durable. Most patients relapse within two years of therapy often due to acquisition of an additional mutation in EGFR kinase domain that confers resistance to TKIs. Crucially, oncogenic EGFR harboring both resistance mutations, T790M and C797S, can no longer be inhibited by currently approved EGFR TKIs. Here, we describe the discovery of BI-4020, which is a noncovalent, wild-type EGFR sparing, macrocyclic TKI. BI-4020 potently inhibits the above-described EGFR variants and induces tumor regressions in a cross-resistant EGFRdel19 T790M C797S xenograft model. Key was the identification of a highly selective but moderately potent benzimidazole followed by complete rigidification of the molecule through macrocyclization.

Discovery and Structure-Based Optimization of Benzimidazole-Derived Activators of SOS1-Mediated Nucleotide Exchange on RAS.

Son of sevenless homologue 1 (SOS1) is a guanine nucleotide exchange factor that catalyzes the exchange of GDP for GTP on RAS. In its active form, GTP-bound RAS is responsible for numerous critical cellular processes. Aberrant RAS activity is involved in ∼30% of all human cancers; hence, SOS1 is an attractive therapeutic target for its role in modulating RAS activation. Here, we describe a new series of benzimidazole-derived SOS1 agonists. Using structure-guided design, we discovered small molecules that increase nucleotide exchange on RAS in vitro at submicromolar concentrations, bind to SOS1 with low double-digit nanomolar affinity, rapidly enhance cellular RAS-GTP levels, and invoke biphasic signaling changes in phosphorylation of ERK 1/2. These compounds represent the most potent series of SOS1 agonists reported to date.

The novel BET bromodomain inhibitor BI 894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML.

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Endovascular Treatment of Occlusive Lesions in the Aortic Bifurcation with Kissing Polytetrafluoroethylene-Covered Stents.

PURPOSE: To determine the clinical outcomes of polytetrafluoroethylene covered balloon expandable stents (CBESs) in occlusive lesions of the aortic bifurcation in a kissing stent configuration. MATERIALS AND METHODS: The study included 69 consecutive patients (29 men, 40 women) who underwent kissing stent procedures with CBESs between January 2003 and April 2009 in a single center. Patients who were previously treated with a CBES were excluded. Follow-up consisted of clinical investigation and duplex ultrasound examination. RESULTS: The primary patency was 88.1% at 1 year and 71.5% at 4 years, with secondary patency rates of 88.1% and 75.3%, respectively. For patients receiving a stent for the first time, primary patency was 91.3% at 1 year and 77.1% at 4 years. For patients who had received previous stents, patency was 83.6% at 1 year and 65.2% at 4 years (P = .83). There were no differences in secondary patency and freedom from target lesion reintervention (TLR). Loss of primary patency was mainly caused by stent occlusions (14 cases [78%]). The freedom from TLR at 4 years was 76.8%. CONCLUSIONS: Patency rates and freedom from TLR of CBESs in the kissing stent configuration with up to 4 years of follow-up were satisfying and mainly affected by stent occlusions. Studies focusing on optimizing stent configuration and medical care to reduce the incidence of thrombosis are indicated to improve results further.

Bispyrimidines as potent histamine H(4) receptor ligands: delineation of structure-activity relationships and detailed H(4) receptor binding mode.

The basic methylpiperazine moiety is considered a necessary substructure for high histamine H4 receptor (H4R) affinity. This moiety is however also the metabolic hot spot for various classes of H4R ligands (e.g., indolcarboxamides and pyrimidines). We set out to investigate whether mildly basic 2-aminopyrimidines in combination with the appropriate linker can serve as a replacement for the methylpiperazine moiety. In the series of 2-aminopyrimidines, the introduction of an additional 2-aminopyrimidine moiety in combination with the appropriate linker lead to bispyrimidines displaying pKi values for binding the human H4R up to 8.2. Furthermore, the methylpiperazine replacement results in compounds with improved metabolic properties. The attempt to transfer the knowledge generated in the class of bispyrimidines to the indolecarboxamides failed. Combining the derived structure-activity relationships with homology modeling leads to new detailed insights in the molecular aspects of ligand-H4R binding in general and the binding mode of the described bispyrimidines in specific.

Detailed structure-activity relationship of indolecarboxamides as H4 receptor ligands.

A series of 76 derivatives of the indolecarboxamide 1 were synthesized, which allows a detailed SAR investigation of this well known scaffold. The data enable the definition of a predictive QSAR model which identifies several compounds with an activity comparable to 1. A selection of these new H(4)R antagonists was synthesized and a comparison of predicted and measured values demonstrates the robustness of the model (47-55). In addition to the H(4)-receptor activity general CMC and DMPK properties were investigated. Some of the new analogs are not only excellently soluble, but display a significantly increased half-life in mouse liver microsomes as well. These properties qualify these compounds as a possible new standard for future in vivo studies (e.g 51, 52 and 55). Moreover, the current studies also provide valuable information on the potential receptor ligand interactions between the indolcarboxamides and the H(4)R protein.

Discovery of orally available integrin alpha5beta1 antagonists.

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin alpha5beta1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin alpha5beta1 inhibitor scaffolds for potential systemic treatment is described.

Novel small molecule bradykinin B1 receptor antagonists. Part 3: hydroxyurea derivatives.

Hydroxy urea moieties are introduced as a new class of bradykinin B(1) receptor antagonists. First, the SAR of the lead compound was systematically explored. Subsequent optimization resulted in the identification of several biaryl-based hydroxyurea bradykinin B(1) receptor antagonists with low-nanomolar activity and very high oral bioavailability in the rat.

Novel small molecule bradykinin B1 receptor antagonists. Part 1: benzamides and semicarbazides.

The synthesis and SAR of two series of bradykinin B(1) receptor antagonists is described. The benzamide moiety proved to be a suitable replacement for the aryl ester functionality of biaryl based antagonists. In addition, it was found that semicarbazides can effectively replace cyclopropyl amino acids. The compounds with the best overall profile were biaryl semicarbazides which display high antagonistic activity, low Caco-2 efflux and high oral bioavailability in the rat.

Novel small molecule bradykinin B1 receptor antagonists. Part 2: 5-membered diaminoheterocycles.

Efforts to find new bradykinin B(1) receptor antagonists identified 2-aminobenzimidazole as a novel core. Subsequent transformation into five-membered diaminoheterocycle derivatives and their synthesis and SAR is described. This resulted in compounds with low nanomolar activity.

SAR of N-phenyl piperidine based oral integrin alpha5beta1 antagonists.

Recently, a new class of selective integrin alpha5beta1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.

Novel small molecule bradykinin B2 receptor antagonists.

Blockade of the bradykinin B(2) receptor provides therapeutic benefit in hereditary angioedema (HAE) and potentially in many other diseases. Herein, we describe the development of highly potent B(2) receptor antagonists with a molecular weight of approximately 500 g/mol. First, known quinoline-based B(2) receptor antagonists were stripped down to their shared core motif 53, which turned out to be the minimum pharmacophore. Targeted modifications of 53 resulted in the highly water-soluble lead compound 8a. Extensive exploration of its structure-activity relationship resulted in a series of highly potent B(2) receptor antagonists, featuring a hydrogen bond accepting functionality, which presumably interacts with the side chain of Asn-107 of the B(2) receptor. Optimization of the microsomal stability and cytochrome P450 inhibition eventually led to the discovery of the highly potent and orally available B(2) receptor antagonist 52e (JSM10292), which showed the best overall properties.

Peptidomimetic C5a receptor antagonists with hydrophobic substitutions at the C-terminus: increased receptor specificity and in vivo activity.

A new class of peptidomimetic C5a receptor antagonists characterized by C-terminal amino acids with hydrophobic side chains is presented. Systematic optimization of the first hits led to JPE1375 (36), which was intensively characterized in vitro and in vivo. Compound 36 exhibits high microsomal stability and receptor specificity and is highly active in an immune complex mediated peritonitis model (reverse passive Arthus reaction) in mice.

Thrombogenicity and related biological properties of heparin bonded collagen coated polyester and human umbilical vein prosthetic vascular grafts.

BACKGROUND: Multiple factors contribute to the process of prosthetic graft failure. Some of them are specifically related to the biological behavior of the used materials. To pursue the ideal substitute for the autologous vein graft, many materials have been taken into consideration. Of these, polyester (Dacron) and human umbilical vein (HUV, Dardik) bypass grafts have gained much attention in vascular surgical practice over the years. This study compares the results of both in vivo and in vitro investigations on graft thrombogenicity and neo-intimal formation in collagen-coated heparin bonded Dacron and in HUV bypass grafts. It is an adjunct to our clinical comparison of graft materials in infrainguinal arterial reconstruction. METHODS: In 12 adult Beagle dogs, a patch was sewn onto the abdominal aorta (Dacron, n = 6; HUV, n = 6). At defined interval times, thrombocyte aggregation was measured with nuclear imaging of 99mTechnetium labeled platelets. Post-mortem histological analysis of the interface between the native vessel wall and the patch was performed in all animals. RESULTS: At 4 h (2.67, SD = 0.77) and after 2 weeks (2.21, SD = 0.28) after implantation, significantly higher thrombogenicity was measured in the HUV grafts compared to Dacron grafts (1.98, SD = 0.10 and 1.98, SD = 0.11, P = 0.02 and 0.025, respectively). At 4 weeks, no significant difference could be found (HUV, 2.26; SD = 0.29; Dacron, 2.11; SD = 0.16; P = 0.23). Measurement of 'neo-intimal' thickness after explantation of the patch at 28 days after the initial procedure showed a significant difference: in HUV grafts the mean thickness of the inner lining was 0.76 mm (SD = 0.50), compared to 0.16 mm (SD = 0.10) in the Dacron grafts (P = 0.013). CONCLUSION: HUV grafts showed a higher thrombogenicity at 4 h and 2 weeks after insertion of the graft compared to Dacron grafts. At 4 weeks this difference is not present. After 28 days the inner ('neo-intimal') lining is significantly more pronounced in HUV grafts than in Dacron grafts.

Borane-ammonia complexes stabilized by hydrogen bonding.

Novel boron-ammonia complexes, wherein an NH(3) molecule is tightly bound through all four of its atoms, have been prepared and studied. The solid-state structure of ortho MOM-phenyllithium is reported. [reaction: see text]

Assessment of prosthetic vascular graft thrombogenicity using the technetium-99m labeled glycoprotein IIb/IIIa receptor antagonist DMP444 in a dog model.

INTRODUCTION: Prosthetic graft patency greatly depends on graft thrombogenicity. The concept of graft thrombogenicity is poorly understood and difficult to measure or quantify. In a study we tested the experimental radiopharmaceutical DMP444 and developed a suitable dog model. This agent is a radiolabelled ((99m)Technetium) glycoprotein IIb/IIIa receptor antagonist with a high affinity for activated platelets. It binds to platelets that are intimately involved in thrombus formation. The agent does not affect thrombocyte function, when used in a dose necessary for imaging. DMP444 does not require platelet harvesting and processing. Early imaging of thrombocyte aggregation sites such as vascular prostheses is possible within 4 hours after injection. MATERIAL AND METHODS: Adult Beagle dogs weighing 12-15 kg were used for the experiments. In 16 dogs a prosthetic patch was sewn onto the abdominal aorta (Bovine pericard: n=4, Dacron: n=6, Human Umbilical Vein: n=6). Imaging cycles after injection of (99m)Technetium-labelled DMP444 were performed on days 1, 7, 14 and 28 after surgery. RESULTS: We noticed differences in thrombus formation on the tested graft materials. The bovine pericard patches (n=4) showed a relatively high rate of thrombocyte aggregation. In the Dacron patches (n=6) aggregation was not seen. In 1 of 6 cases of human umbilical vein patches a measurable focal aggregation was recorded. CONCLUSION: The method outlined in this study is a relatively simple and reproducable method to visualize thrombocyte aggregation.