A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

The Effect of Core Replacement on S64315, a Selective MCL-1 Inhibitor, and Its Analogues.

Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution.

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-µM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.

Structure-guided design of alpha-amino acid-derived Pin1 inhibitors.

The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of alpha-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC(50) <100nM, but were inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10-50-fold loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked proliferation of PC3 cells.

Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design.

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.

Identification of chemically diverse Chk1 inhibitors by receptor-based virtual screening.

Inhibition of the Chk1 kinase by small molecules is of great therapeutic interest for oncology and in understanding the cellular regulation of the G2/M checkpoint. We report how computational docking of a large electronic catalogue of compounds to an X-ray structure of the Chk1 ATP-binding site allowed prioritisation of a small subset of these compounds for assay. This led to the discovery of 10 novel Chk1 inhibitors, distributed among nine new and clearly different chemical scaffolds. Several of these scaffolds have promising lead-like properties. All these ligands act by competitive binding to the targeted ATP site. The crystal structures of four of these compounds bound to this site are presented, and reasonable modelled docking modes are suggested for the 5 other scaffolds. This structural context is used to assess the potential of these scaffolds for further medicinal chemistry efforts, suggesting that several of them could be elaborated to make additional interactions with the buried part of the ATP site. Some unusual interactions with the conserved kinase backbone motif are pointed out. The ligand-binding modes are also used to discuss their medicinal chemistry potential with respect to undesirable chemical functionalities, whether these functionalities bind directly to the protein or not. Overall, this work illustrates how virtual screening can identify a diverse set of ligands which bind to the targeted site. The structural models for these ligands in the Chk1 ATP-binding site will facilitate further medicinal chemistry efforts targeting this kinase.

Triazolo[1,5-a]pyrimidines as novel CDK2 inhibitors: protein structure-guided design and SAR.

Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.

Structure-based design of novel Chk1 inhibitors: insights into hydrogen bonding and protein-ligand affinity.

We report the discovery, synthesis, and crystallographic binding mode of novel furanopyrimidine and pyrrolopyrimidine inhibitors of the Chk1 kinase, an oncology target. These inhibitors are synthetically tractable and inhibit Chk1 by competing for its ATP site. A chronological account allows an objective comparison of modeled compound docking modes to the subsequently obtained crystal structures. The comparison provides insights regarding the interpretation of modeling results, in relationship to the multiple reasonable docking modes which may be obtained in a kinase-ATP site. The crystal structures were used to guide medicinal chemistry efforts. This led to a thorough characterization of a pair of ligand-protein complexes which differ by a single hydrogen bond. An analysis indicates that this hydrogen bond is expected to contribute a fraction of the 10-fold change in binding affinity, adding a valuable observation to the debate about the energetic role of hydrogen bonding in molecular recognition.

Structure-guided design of pyrazolo[1,5-a]pyrimidines as inhibitors of human cyclin-dependent kinase 2.

The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.