Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA2).

Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

Structure of the Epigenetic Oncogene MMSET and Inhibition by N-Alkyl Sinefungin Derivatives.

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.

Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered through X-ray Fragment Screening.

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors.

The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen against DDR1 and identified fragments that bound either at the hinge or in the back pocket associated with the DFG-out conformation of the kinase. Modeling based on crystal structures of potent kinase inhibitors facilitated the "back-to-front" design of potent DDR1/2 inhibitors that incorporated one of the DFG-out fragments. Further optimization led to low nanomolar, orally bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell lines.

Perspective on computational and structural aspects of kinase discovery from IPK2014.

Recent advances in understanding the activity and selectivity of kinase inhibitors and their relationships to protein structure are presented. Conformational selection in kinases is studied from empirical, data-driven and simulation approaches. Ligand binding and its affinity are, in many cases, determined by the predetermined active and inactive conformation of kinases. Binding affinity and selectivity predictions highlight the current state of the art and advances in computational chemistry as it applies to kinase inhibitor discovery. Kinome wide inhibitor profiling and cell panel profiling lead to a better understanding of selectivity and allow for target validation and patient tailoring hypotheses. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase.

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 µM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.

Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK.

Fragment-based approaches to the discovery of kinase inhibitors.

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Crystal structure of human soluble adenylate cyclase reveals a distinct, highly flexible allosteric bicarbonate binding pocket.

Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,ß-methylene adenosine 5'-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.

Potent, selective inhibitors of fibroblast growth factor receptor define fibroblast growth factor dependence in preclinical cancer models.

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.

Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor.

A novel inhibitor of Schistosoma PNP was identified using an "in silico" approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC(50) of 1.3 microM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.

Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor with potent aurora kinase activity.

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

Identification of N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxamide (AT7519), a novel cyclin dependent kinase inhibitor using fragment-based X-ray crystallography and structure based drug design.

The application of fragment-based screening techniques to cyclin dependent kinase 2 (CDK2) identified multiple (>30) efficient, synthetically tractable small molecule hits for further optimization. Structure-based design approaches led to the identification of multiple lead series, which retained the key interactions of the initial binding fragments and additionally explored other areas of the ATP binding site. The majority of this paper details the structure-guided optimization of indazole (6) using information gained from multiple ligand-CDK2 cocrystal structures. Identification of key binding features for this class of compounds resulted in a series of molecules with low nM affinity for CDK2. Optimisation of cellular activity and characterization of pharmacokinetic properties led to the identification of 33 (AT7519), which is currently being evaluated in clinical trials for the treatment of human cancers.

Identification of inhibitors of protein kinase B using fragment-based lead discovery.

Using fragment-based screening techniques, 5-methyl-4-phenyl-1H-pyrazole (IC50 80 microM) was identified as a novel, low molecular weight inhibitor of protein kinase B (PKB). Herein we describe the rapid elaboration of highly potent and ligand efficient analogues using a fragment growing approach. Iterative structure-based design was supported by protein-ligand structure determinations using a PKA-PKB "chimera" and a final protein-ligand structure of a lead compound in PKBbeta itself.

Rapid evolution of 6-phenylpurine inhibitors of protein kinase B through structure-based design.

6-phenylpurines were identified as novel, ATP-competitive inhibitors of protein kinase B (PKB/Akt) from a fragment-based screen and were rapidly progressed to potent compounds using iterative protein-ligand crystallography with a PKA-PKB chimeric protein. An elaborated lead compound showed cell growth inhibition and effects on cellular signaling pathways characteristic of PKB inhibition.

Structure-based design of isoquinoline-5-sulfonamide inhibitors of protein kinase B.

Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells.