Peter Sampson

Methionine In and Out of Proteins: Targets for Drug Design

The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets. Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics. As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs. Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design. The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes. This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted.

The Discovery of PLK4 Inhibitors: (E)-3-((1H-Indazol-6-yl)methylene)indolin-2-ones as Novel Antiproliferative Agents

The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.

Synthesis of an α-aminophosphonate nucleoside as an inhibitor of S-adenosyl-L-homocysteine hydrolase

A phosphonic acid analogue of S-adenosyl-L-homocysteine was prepared by a novel method and the epimeric mixture separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase, however each presented distinct kinetic characteristics.

2,3,4,5-Tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines as inhibitors of the bacterial enoyl ACP reductase, FabI

In the search for new antibacterial agents, the enzyme FabI has been identified as an attractive target. Employing a structure guided approach, the previously reported ene-amide series of FabI inhibitors were expanded to include 2,3,4,5-tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines. These novel series incorporate additional H-bonding functions and can be more water soluble than their naphthyridinone progenitors; diazepine 16c is shown to be efficacious in a mouse infection model.

The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 μM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.

Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Spiro-naphthyridinone piperidines as inhibitors of S. aureus and E. coli enoyl-ACP reductase (FabI).

Spiropiperidine naphthyridinone inhibitors of Staphylococcus aureus and Escherichia coli FabI have been prepared. Compounds 14a and 14c were identified as having sub-nanomolar E. coli FabI activity and are among the most potent FabI inhibitors yet described. The structural model of 14a bound to E. coli FabI is shown.

Syntheses and kinetic evaluation of hydroxamate-based peptide inhibitors of glyoxalase I

Hydroxamate-containing tripeptide analogs resembling a reactive intermediate in glyoxalase I catalysis were prepared by solution methods and were found to be competitive inhibitors of the enzyme from Saccharomyces cerevisiae. Electronic properties of the hydroxamate functionality as well as those of the expected intermediates in the enzyme-catalyzed reaction were compared.

Abstract CT066: First-in-human phase I trial of the oral PLK4 inhibitor CFI-400945 in patients with advanced solid tumors

Background: CFI-400945 is a first-in-class, potent, selective, orally active inhibitor of Polo-like kinase 4 (PLK4) (Ki = 0.26 nM), a conserved master regulator of centriole duplication that is important for maintenance of genomic integrity. In preclinical studies, CFI-400945 demonstrated robust anti-tumor activity, including durable complete tumor regression, in a large number of patient-derived xenograft models from multiple tumor types (breast, ovarian, pancreas, prostate carcinomas; some derived from heavily pre-treated patients). The objectives of this first-in-human phase I trial are to establish safety, tolerability, pharmacokinetics, and recommended phase II dose (RP2D) of CFI-400945. Methods: Patients with advanced solid tumors age >18 years, with ECOG performance status of 0-1, adequate organ function and RECIST-measurable disease progressing on standard treatments are eligible. Dose escalation follows a standard 3+3 design, with a starting dose of 3mg once daily continuous oral dosing based upon preclinical data and a severely toxic dose to 10 percent (STD10) in rats of 3 mg/kg. The primary endpoint is the incidence of dose limiting toxicities (DLTs) during the first cycle. Safety assessments using CTCAE version 4.03 criteria are performed weekly during the first three cycles and then every two weeks. Response assessments are performed every two cycles. Results: From April/14 to December/15, 31 patients were enrolled across eight pre-defined dose levels (3, 6, 11, 16, 24, 32, 48 and 72 mg). No DLT events have been observed. Dose escalation at 96mg is currently ongoing. The most frequent treatment-related adverse events (trAEs) include fatigue (24%), diarrhea (17.2%), nausea (17.2%), decreased appetite (13.8%) and vomiting (6.9%). All trAEs were grade 1 or grade 2. Fifteen serious adverse events (SAEs) have occurred in 9 patients, all considered unrelated to CFI-400945 treatment. Preliminary PK results estimate a half-life of about 10 hours, with Cmax and AUC showing dose proportionality. Two patients enrolled at the 48 mg dose level have completed >6 cycles, including a patient with KRAS mutant colorectal cancer who achieved 24% reduction in target lesions and >50% reduction in serum CEA levels. Conclusions: CFI-400945 is well tolerated at doses up to 72mg with a favorable PK profile. Preliminary evidence of anti-tumor activity has been observed. Exploration of 96mg daily dosing is ongoing and once the RP2D has been established exploration of anti-tumor activity in biomarker-driven expansion arms of CFI-400945 in indications including advanced breast cancer is planned. Updated results of this ongoing trial will be presented at the meeting. Citation Format: Philippe L. Bedard, David W. Cescon, Graham Fletcher, Trish Denny, Richard Brokx, Peter Sampson, Mark R. Bray, Dennis J. Slamon, Tak W. Mak, Zev A. Wainberg. First-in-human phase I trial of the oral PLK4 inhibitor CFI-400945 in patients with advanced solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT066.