Identification of a selective inhibitor of transforming growth factor ß-activated kinase 1 by biosensor-based screening of focused libraries.

Transforming growth factor-ß activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, plays an essential role in mediating signals from various pro-inflammatory cytokines and therefore may be a good target for developing anti-inflammation agents. Herein, we report our efforts to identify TAK1 inhibitors with a good selectivity profile with which to initiate medicinal chemistry. Instead of resorting to a high-throughput screening campaign, we performed biosensor-based biophysical screening for a limited number of compounds by taking advantage of existing knowledge on kinase inhibitors. Rather than focusing on one specific inhibition mode, we searched for three different types, Type I (ATP-competitive, DFG-in), Type II (DFG-out), and Type III binders (non-ATP competitive) in parallel, and succeeded in identifying candidates in all three categories efficiently and rapidly. Finally, the biosensor-based binding kinetics for the active and inactive forms of TAK1 were measured to prioritize the Type I and Type II inhibitors. The effort resulted in the identification of a new TAK1-selective Type I compound with a thienopyrimidine scaffold that served as a good starting point for medicinal chemistry.

Discovery of a potent and highly selective transforming growth factor ß receptor-associated kinase 1 (TAK1) inhibitor by structure based drug design (SBDD).

A novel thienopyrimidinone analog was discovered as a potent and highly selective TAK1 inhibitor using the SBDD approach. TAK1 plays a key role in inflammatory and immune signaling, so TAK1 is considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). After the hit compound had been obtained, our modifications successfully increased TAK1 inhibitory activity and solubility, but metabolic stability was still unsatisfactory. To improve metabolic stability, we conducted metabolic identification. Although the obtained metabolite was fortunately a potent TAK1 inhibitor, its kinase selectivity was low. Subsequently, to achieve high kinase selectivity, we used SBDD to follow two strategies: one targeting unique amino acid residues in TAK1, especially the combination of Ser111 and Asn114; the other decreasing the interaction with Tyr106 at the hinge position in TAK1. As expected, our designed compound showed an excellent kinase selectivity profile in both an in-house and a commercially available panel assay of over 420 kinases and also retained its potent TAK1 inhibitory activity (TAK1 IC50=11nM).

Development of a Method for Converting a TAK1 Type I Inhibitor into a Type II or c-Helix-Out Inhibitor by Structure-Based Drug Design (SBDD).

We have developed a method for converting a transforming growth factor-ß-activated kinase 1 (TAK1) type I inhibitor into a type II or c-helix-out inhibitor by structure-based drug design (SBDD) to achieve an effective strategy for developing these different types of kinase inhibitor in parallel. TAK1 plays a key role in inflammatory and immune signaling, and is therefore considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). We have already reported novel type I TAK1 inhibitor, so we utilized its X-ray information to design a new chemical class type II and c-helix-out inhibitors. To develop the type II inhibitor, we superimposed the X-ray structure of our reported type I inhibitor onto a type II compound that inhibits multiple kinases, and used SBDD to design a new type II inhibitor. For the TAK1 c-helix-out inhibitor, we utilized the X-ray structure of a b-Raf c-helix-out inhibitor to design compounds, because TAK1 is located close to b-Raf in the Sugen kinase tree, so we considered that TAK1 would, similarly to b-Raf, form a c-helix-out conformation. The X-ray crystal structure of the inhibitors in complex with TAK1 confirmed the binding modes of the compounds we designed. This report is notable for being the first discovery of a c-helix-out inhibitor against TAK1.

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors.

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (Kd=0.52nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC50=0.098µM, NCI-N87 IC50=0.066µM) and also displayed high oral bioavailability in mice (F=44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition=136%).

Preclinical antitumor activity of the novel heat shock protein 90 inhibitor CH5164840 against human epidermal growth factor receptor 2 (HER2)-overexpressing cancers.

Heat shock protein 90 (Hsp90), a molecular chaperone that plays a significant role in the stability and maturation of client proteins, including oncogenic targets for cell transformation, proliferation, and survival, is an attractive target for cancer therapy. We identified the novel Hsp90 inhibitor, CH5164840, and investigated its induction of oncogenic client protein degradation, antiproliferative activity, and apoptosis against an NCI-N87 gastric cancer cell line and a BT-474 breast cancer cell line. Interestingly, CH5164840 demonstrated tumor selectivity both in vitro and in vivo, binding to tumor Hsp90 (which forms active multiple chaperone complexes) in vitro, and being distributed effectively to tumors in a mouse model, which, taken together, supports the decreased levels of phosphorylated Akt by CH5164840 that we observed in tumor tissues, but not in normal tissues. As well as being well tolerated, the oral administration of CH5164840 exhibited potent antitumor efficacy with regression in NCI-N87 and BT-474 tumor xenograft models. In addition, CH5164840 significantly enhanced antitumor efficacy against gastric and breast cancer models when combined with the human epidermal growth factor receptor 2 (HER2)-targeted agents, trastuzumab and lapatinib. These data demonstrate the potent antitumor efficacy of CH5164840 when administered alone, and its significant combination efficacy when combined with trastuzumab or lapatinib, supporting the clinical development of CH5164840 as an Hsp90 inhibitor for combination therapy with HER2-targeted agents against HER2-overexpressing tumors.

Design and evaluation of azaindole-substituted N-hydroxypyridones as glyoxalase I inhibitors.

We conducted a high throughput screening for glyoxalase I (GLO1) inhibitors and identified 4,6-diphenyl-N-hydroxypyridone as a lead compound. Using a binding model of the lead and public X-ray coordinates of GLO1 enzymes complexed with glutathione analogues, we designed 4-(7-azaindole)-substituted 6-phenyl-N-hydroxypyridones. 7-Azaindole's 7-nitrogen was expected to interact with a water network, resulting in an interaction with the protein. We validated this inhibitor design by comparing its structure-activity relationship (SAR) with that of corresponding indole derivatives, by analyzing the binding mode with X-ray crystallography and by evaluating its thermodynamic binding parameters.

Design and synthesis of novel macrocyclic 2-amino-6-arylpyrimidine Hsp90 inhibitors.

Macrocyclic compounds bearing a 2-amino-6-arylpyrimidine moiety were identified as potent heat shock protein 90 (Hsp90) inhibitors by modification of 2-amino-6-aryltriazine derivative (CH5015765). We employed a macrocyclic structure as a skeleton of new inhibitors to mimic the geldanamycin-Hsp90 interactions. Among the identified inhibitors, CH5164840 showed high binding affinity for N-terminal Hsp90α (K(d)=0.52nM) and strong anti-proliferative activity against human cancer cell lines (HCT116 IC(50)=0.15µM, NCI-N87 IC(50)=0.066µM). CH5164840 displayed high oral bioavailability in mice (F=70.8%) and potent antitumor efficacy in a HCT116 human colorectal cancer xenograft model (tumor growth inhibition=83%).

Lead generation of heat shock protein 90 inhibitors by a combination of fragment-based approach, virtual screening, and structure-based drug design.

Heat shock protein 90 (Hsp90) is a molecular chaperone which regulates maturation and stabilization of its substrate proteins, known as client proteins. Many client proteins of Hsp90 are involved in tumor progression and survival and therefore Hsp90 can be a good target for developing anticancer drugs. With the aim of efficiently identifying a new class of orally available inhibitors of the ATP binding site of this protein, we conducted fragment screening and virtual screening in parallel against Hsp90. This approach quickly identified 2-aminotriazine and 2-aminopyrimidine derivatives as specific ligands to Hsp90 with high ligand efficiency. In silico evaluation of the 3D X-ray Hsp90 complex structures of the identified hits allowed us to promptly design CH5015765, which showed high affinity for Hsp90 and antitumor activity in human cancer xenograft mouse models.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Autonomous motion of vesicle via ion exchange.

The autonomous motion of vesicle is observed in a simple chemical system. A vesicle composed of didodecyldimethylammonium bromide DDAB breaks down by ion exchange from Br(-) to I(-). When an electrolyte is supplied to vesicles, some of them begin to move after an induction period. They continue to move, leaving behind the reaction products on the trail. The ion exchange decreases the vesicle size, and smaller vesicles remain after the motion. We examine the characteristics of this motion. The surface tension of the DDAB-containing aqueous phase depends on the KI concentration. Considering this result carefully, we conclude that vesicles can move when the ion exchange from Br(-) to I(-) proceeds irreversibly. Then, inhomogeneity in the vesicle membrane develops because of the coagulating nature of the product, didodecyldimethylammonium iodide (DDAI), which is sparingly soluble in water. Inhomogeneous properties of vesicle membranes are then generated, which induce surface transport of the reaction product and flow in the water pool. As a result, a couple of convection rolls appear in the water pool of the vesicle. The convection rolls drive vesicle motion. A simple model for the semiquantitative description is proposed.

[Fragment screening using surface plasmon resonance optical biosensor technology].

A surface plasmon resonance (SPR) optical biosensor is a label-free biophysical device which can detect molecular interaction in real time. SPR is an emerging technology for fragment screening, the first step in fragment-based drug discovery. Low levels of protein consumption and the ability to detect interactions with K(d) as low as mM make this technology particularly attractive. Inherently small SPR responses due to fragment binding had been an issue but, owing to well-established experimental protocols, such responses have become readily detectable. Medium-throughput instruments are now on the market from several manufacturers that enable complete screening of a library with several thousand compounds within a few days. In fragment screening, test compounds are injected at a high concentration because of the low affinity expected for small molecules, making it likely that many false positives arise from non-specific binding to an unrelated part of the target protein. Such false positives have to be eliminated by a well-designed assay cascade so as to select true hits which can then be subjected to X-ray crystallization to obtain detailed structural information. SPR-based direct binding assays have to be developed with a sufficient binding capacity and good reproducibility with a Z'-factor larger than 0.6. In selecting hit candidates from fragment primary screens, the shape of sensorgrams, binding stoichiometry and response level to reference proteins when available must be carefully evaluated. The selected compounds from primary screening need to be further examined in terms of dose-dependence and binding competition against tight binding reference compounds to ensure that they bind to the designated site of the target protein.

Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases.

Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.

Establishment of standard values for the latency, interval and amplitude parameters of tibial nerve somatosensory evoked potentials (SEPs).

OBJECTIVE: To establish standard values for tibial nerve somatosensory evoked potentials (SEPs). METHODS: We examined SEPs following left tibial nerve stimulation in 65 normal subjects of various ages, and performed multiple regression analysis using height, age, (age-20)(2) and gender as predictor variables. We objectively selected the latency or interval parameters with less intersubject variability as the standard parameters for evaluation. RESULTS: Among 3 cortical bipolar derivations investigated, the Cz'-Cc lead gave a more constant and stable P38 component than the Cz'-Fz or Ci-Cc lead. The latencies of the N8o (N8 onset) of the popliteal potential, P15 (P15 peak) in the contralateral iliac crest-ipsilateral greater trochanter lead, N21, N30 and P38o/P38 in the Cz'-Cc lead, as well as the intervals between these components were selected as standard parameters. P15 was easily identified in all of the subjects and is expected to be a new parameter to evaluate the proximal segment of the tibial nerve. The amplitudes of P15 and the other components were also evaluated. We present nomograms for the normal limit values of each parameter. CONCLUSIONS: We present a thorough set of standard values for tibial SEPs where the subject factors were fully considered, and which is easily applicable to clinical practice.

[An adult case of Moyamoya disease presenting with transient hemichorea].

A 54-year-old woman presented choreic movements in left face and extremities for three months. Cerebral angiography revealed occlusions of bilateral internal carotid arteries (right: after the furcation of posterior communicating artery; left: after the furcation of opthalmic artery) and net-like collaterals around the basal ganglia region (Moyamoya vessels). Haloperidol rapidly resolved the choreic movements and no recurrence was observed. PET demonstrated misery perfusion at bilateral temporo-parietal cortices. Especially, right peri-sylvian region showed the most severe ischemia. MRI demonstrated no infarcts. Therefore, ischemia of the right striatum was suspected to be the cause of the left-sided hemichorea. Previously, Moyamoya disease presenting chorea was infrequently reported in young people of less than 20 years of age. This is the first report of the aged patient of Moyamoya disease presenting with hemichorea and severe misery perfusion.

Disruption of CRAF-mediated MEK activation is required for effective MEK inhibition in KRAS mutant tumors.

MEK inhibitors are clinically active in BRAF(V600E) melanomas but only marginally so in KRAS mutant tumors. Here, we found that MEK inhibitors suppress ERK signaling more potently in BRAF(V600E), than in KRAS mutant tumors. To understand this, we performed an RNAi screen in a KRAS mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAF(V600E). MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF.

Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.

Tumors with mutant RAS are often dependent on extracellular signal-regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.