Structures and mechanism of human glycosyltransferase ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), an important player in immune homeostasis.

ß1,3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi-resident glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. They catalyze the addition of the N-acetylglucosamine to the N-acetyl-lactosamine repeat as a key step of the chain elongation process. Poly-N-acetyl-lactosamine is involved in the immune system in many ways. Particularly, its long chain has been demonstrated to suppress excessive immune responses. Among the characterized B3GNTs, B3GNT2 is the major poly-N-acetyl-lactosamine synthase, and deletion of its coding gene dramatically reduced the cell surface poly-N-acetyl-lactosamine and led to hypersensitive and hyperresponsive immunocytes. Despite the extensive functional studies, no structural information is available to understand the molecular mechanism of B3GNT2, as well as other B3GNTs. Here we present the structural and kinetic studies of the human B3GNT2. Five crystal structures of B3GNT2 have been determined in the unliganded, donor substrate-bound, acceptor substrate-bound, and product(s)-bound states at resolutions ranging from 1.85 to 2.35 Å. Kinetic study shows that the transglycosylation reaction follows a sequential mechanism. Critical residues involved in recognition of both donor and acceptor substrates as well as catalysis are identified. Mutations of these invariant residues impair B3GNT2 activity in cell assays. Structural comparison with other glycosyltransferases such as mouse Fringe reveals a novel N-terminal helical domain of B3GNTs that may stabilize the catalytic domain and distinguish among different acceptor substrates.

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 µM in the LAD2 cell assay.

Optimization beyond AMG 232: discovery and SAR of sulfonamides on a piperidinone scaffold as potent inhibitors of the MDM2-p53 protein-protein interaction.

We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.

Creating a more strategic small molecule biophysical hit characterization workflow.

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Imidazolone as an Amide Bioisostere in the Development of ß-1,3-N-Acetylglucosaminyltransferase 2 (B3GNT2) Inhibitors.

B3GNT2 is responsible for elongation of cell surface long-chain polylactosamine, which influences the regulation of the immune response, making it an attractive target for immunomodulation. In the development of amide containing B3GNT2 inhibitors guided by structure-based drug design, imidazolones were found to successfully serve as amide bioisosteres. This novel imidazolone isosteric strategy alleviated torsional strain of the amide bond on binding to B3GNT2 and improved potency, isoform selectivity, as well as certain physicochemical and pharmacokinetic properties. Herein, we present the synthesis, SAR, X-ray cocrystal structures, and in vivo PK properties of imidazol-4-ones in the context of B3GNT2 inhibition.

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors.

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.

An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics.

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration-response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease.

Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 (PNPLA3) with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While Pnpla3-deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type PNPLA3 in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous PNPLA3 rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA). Limiting our search to SNP-spanning triggers, a series of chemically modified siRNA were tested in vitro for activity and selectivity toward PNPLA3 rs738409 mRNA. Conjugation of the siRNA to a triantennary N-acetylgalactosamine (GalNAc) ligand enabled in vivo screening using adeno-associated virus to overexpress human PNPLA3I148M versus human PNPLA3I148I in mouse livers. Structure-activity relationship optimization yielded potent and minor allele-specific compounds that achieved high levels of mRNA and protein knockdown of human PNPLA3I148M but not PNPLA3I148I. Testing of the minor allele-specific siRNA in PNPLA3I148M-expressing mice fed a NASH-inducing diet prevented PNPLA3I148M-driven disease phenotypes, thus demonstrating the potential of a precision medicine approach to treating NAFLD.

Discovery and initial optimization of 5,5'-disubstituted aminohydantoins as potent beta-secretase (BACE1) inhibitors.

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S(1) to the S(3) pocket.

Discovery and in Vivo Evaluation of Macrocyclic Mcl-1 Inhibitors Featuring an α-Hydroxy Phenylacetic Acid Pharmacophore or Bioisostere.

Overexpression of the antiapoptotic protein Mcl-1 provides a survival advantage to some cancer cells, making inhibition of this protein an attractive therapeutic target for the treatment of certain types of tumors. Herein, we report our efforts toward the identification of a novel series of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. This work led to the discovery of 1, a potent Mcl-1 inhibitor (IC50 = 19 nM in an OPM-2 cell viability assay) with good pharmacokinetic properties and excellent in vivo efficacy in an OPM-2 multiple myeloma xenograft model.

A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase.

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20 nM acetyl-CoA, compared to 3 muM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening and yields excellent assay statistics (Z' > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that generate acetyl-CoA.

A "Click Chemistry Platform" for the Rapid Synthesis of Bispecific Molecules for Inducing Protein Degradation.

Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.

Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses.

Discovery of AM-7209, a potent and selective 4-amidobenzoic acid inhibitor of the MDM2-p53 interaction.

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Novel inhibitors of the MDM2-p53 interaction featuring hydrogen bond acceptors as carboxylic acid isosteres.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Discovery of Potent and Simplified Piperidinone-Based Inhibitors of the MDM2-p53 Interaction.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

Discovery of AMG 925, a FLT3 and CDK4 dual kinase inhibitor with preferential affinity for the activated state of FLT3.

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.

Discovery of AMG 232, a potent, selective, and orally bioavailable MDM2-p53 inhibitor in clinical development.

We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).