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KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3–5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking. Treatment of KRASG12C-mutant cancer cells with the KRAS(G12C) inhibitor AMG 510 leads to durable response in mice, and anti-tumour activity in patients suggests that AMG 510 could be effective in patients for whom treatments are currently lacking.

The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Replacement of the piperazine sulfonamide portion of the PI3Kα inhibitor AMG 511 (1) with a range of aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated an EC50 of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing.

Activating mutations in RAS represent the most common oncogenic driver mutation in cancer. The single amino acid substitution of cysteine for glycine at position 12 (KRASG12C) is frequently found in solid malignancies, particularly in lung adenocarcinoma (~13%), colorectal adenocarcinoma (3%), and pancreatic adenocarcinoma (~1%). Recently it has been demonstrated that KRASG12C can be targeted with covalent small molecule inhibitors which react with the mutant cysteine adjacent to the switch II pocket (SIIP), locking KRAS in its inactive GDP-bound state. We describe here the discovery and in vitro characterization of AMG 510, a covalent inhibitor of KRASG12C possessing potent biochemical and cellular activity, as well as robust in vivo efficacy. AMG 510 inhibited SOS1-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. The observed rate constant (kinact/Ki) of covalent modification of KRASG12C by AMG 510 was determined biochemically by mass spectrometry as well as in the cellular context (kobs/[I]). Cysteine proteome analysis of cells treated with AMG 510 revealed that only the G12C-containing peptide of KRAS was covalently modified. AMG 510 inhibited KRAS signaling as measured by ERK phosphorylation in all KRAS p.G12C cell lines tested, but did not inhibit phosphorylation of ERK in cell lines lacking the KRAS p.G12C mutation. Cellular occupancy of KRASG12C by AMG 510 was determined by mass spectrometry and correlated well with inhibition of ERK phosphorylation. AMG 510 also selectively impaired the viability of KRAS p.G12C mutant lines. Combination treatment of AMG 510 with inhibitors of other cellular signaling pathways exhibited evidence for synergistic effects on cell viability. Treatment of KRAS p.G12C lines with covalent KRASG12C inhibitors increased the expression of HLA. To test the impact of KRASG12C inhibition on immune surveillance in vivo, we generated a syngeneic tumor cell line that is suitable for testing AMG 510 in combination with checkpoint inhibitor therapies and characterized this line in vitro. AMG 510 is currently being evaluated in a Phase I study in patients with solid tumors harboring KRAS p.G12Cmutations. Citation Format: Anne Y. Saiki, Kevin Gaida, Karen Rex, Pragathi Achanta, Tisha San Miguel, Neelima Koppada, Dhanashri Bagal, Brian A. Lanman, Robert S. Foti, John D. McCarter, Laurie P. Volak, Jude Canon, Victor J. Cee, J. Russell Lipford. Discovery and in vitro characterization of AMG 510–a potent and selective covalent small-molecule inhibitor of KRASG12C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4484.

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

Phosphoinositide 3-kinase (PI3K) is an important target in oncology due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of 4-amino-6-methyl-1,3,5-triazine sulfonamides were synthesized and evaluated as inhibitors of PI3K. The synthesis, in vitro biological activities, pharmacokinetic and in vivo pharmacodynamic profiling of these compounds are described. The most promising compound from this investigation (compound 3j) was found to be a pan class I PI3K inhibitor with a moderate (>10-fold) selectivity over the mammalian target of rapamycin (mTOR) in the enzyme assay. In a U87 MG cellular assay measuring phosphorylation of Akt, compound 3j displayed low double digit nanomolar IC(50) and exhibited good oral bioavailability in rats (F(oral)=63%). Compound 3j also showed a dose dependent reduction in the phosphorylation of Akt in a U87 tumor pharmacodynamic model with a plasma EC(50)=193 nM (91 ng/mL).

The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 4-(2-((6-methoxypyridin-3-yl)amino)-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (1). In this paper, we describe the optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and was designated AMG 511.

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

β-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aβ40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of β-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS β-amyloid (Aβ) in Sprague-Dawley rats following oral administration.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-β peptide (Aβ) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aβ levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (1) is a potent and efficacious inhibitor of PI3Kα and mTOR in vitro and in vivo. However, in hepatocyte and in vivo metabolism studies, 1 was found to undergo deacetylation on the 2-amino substituent of the benzothiazole. As an approach to reduce or eliminate this metabolic deacetylation, a variety of 6,5-heterocyclic analogues were examined as an alternative to the benzothiazole ring. Imidazopyridazine 10 was found to have similar in vitro potency and in vivo efficacy relative to 1, while only minimal amounts of the corresponding deacetylated metabolite of 10 were observed in hepatocytes.

The phosphoinositide 3-kinase (PI3K) family catalyzes the ATP-dependent phosphorylation of the 3'-hydroxyl group of phosphatidylinositols and plays an important role in cell growth and survival. There is abundant evidence demonstrating that PI3K signaling is dysregulated in many human cancers, suggesting that therapeutics targeting the PI3K pathway may have utility for the treatment of cancer. Our efforts to identify potent, efficacious, and orally available PI3K/mammalian target of rapamycin (mTOR) dual inhibitors resulted in the discovery of a series of substituted quinolines and quinoxalines derivatives. In this report, we describe the structure-activity relationships, selectivity, and pharmacokinetic data of this series and illustrate the in vivo pharmacodynamic and efficacy data for a representative compound.

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inbibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches.

A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC50=0.71 μM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL=18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC50=1.1 nM, pMCM2 IC50=32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.

Cdc7 kinase is responsible for the initiation and regulation of DNA replication and has been proposed as a target for cancer therapy. We have identified a class of Cdc7 inhibitors based on a substituted indole core. Synthesis of focused indole and azaindole analogs yielded potent and selective 5-azaindole Cdc7 inhibitors with improved intrinsic metabolic stability (ie 36). In parallel, quantum mechanical conformational analysis helped to rationalize SAR observations, led to a proposal of the preferred binding conformation in the absence of co-crystallography data, and allowed the design of 7-azaindole 37 as a second lead in this series.

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.

The kinase activity of the serine/threonine kinase STK33 was recently shown to be required for the survival of cancer cell lines dependent on the KRAS oncogene. This suggests that an STK33 kinase inhibitor could be useful to treat cancer patients whose tumors harbor KRAS mutations. Our goal was to identify STK33 kinase inhibitors to explore the role of this kinase in mutant KRAS cancer cell lines. First, full-length wild-type or kinase dead (KD) STK33 proteins were prepared using a baculovirus/insect expression system and purified by chromatography. Preliminary experiments identified two peptides, CREBtide and p70S6K peptides, as potential substrates for STK33. Both peptides were therefore used to track STK33 kinase activity during purification steps. We found that phosphorylation of the p70S6K peptide correlated well with STK33 presence during purification, but the peptide was not phosphorylated by the kinase dead STK33. In contrast, CREBtide phosphorylation showed a poor correlation with STK33 positive fractions and also occurred in the presence of a kinase dead STK33. The p70S6K peptide was thus chosen as the substrate for final optimization of an STK33 kinase assay. Over 400 000 compounds were screened using a high throughput STK33 kinase assay. Multiple hits were identified and IC50s were determined for the top 1043 compounds. Multiple potent STK33 kinase inhibitors were identified, including 69 compounds with IC50s Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 253. doi:10.1158/1538-7445.AM2011-253