Ripretinib (DCC-2618) Is a Switch Control Kinase Inhibitor of a Broad Spectrum of Oncogenic and Drug-Resistant KIT and PDGFRA Variants.

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.

Phase 1 dose-finding study of rebastinib (DCC-2036) in patients with relapsed chronic myeloid leukemia and acute myeloid leukemia.

A vailable tyrosine kinase inhibitors for chronic myeloid leukemia bind in an adenosine 5'-triphosphate-binding pocket and are affected by evolving mutations that confer resistance. Rebastinib was identified as a switch control inhibitor of BCR-ABL1 and FLT3 and may be active against resistant mutations. A Phase 1, first-in-human, single-agent study investigated rebastinib in relapsed or refractory chronic or acute myeloid leukemia. The primary objectives were to investigate the safety of rebastinib and establish the maximum tolerated dose and recommended Phase 2 dose. Fifty-seven patients received treatment with rebastinib. Sixteen patients were treated using powder-in-capsule preparations at doses from 57 mg to 1200 mg daily, and 41 received tablet preparations at doses of 100 mg to 400 mg daily. Dose-limiting toxicities were dysarthria, muscle weakness, and peripheral neuropathy. The maximum tolerated dose was 150 mg tablets administered twice daily. Rebastinib was rapidly absorbed. Bioavailability was 3- to 4-fold greater with formulated tablets compared to unformulated capsules. Eight complete hematologic responses were achieved in 40 evaluable chronic myeloid leukemia patients, 4 of which had a T315I mutation. None of the 5 patients with acute myeloid leukemia responded. Pharmacodynamic analysis showed inhibition of phosphorylation of substrates of BCR-ABL1 or FLT3 by rebastinib. Although clinical activity was observed, clinical benefit was insufficient to justify continued development in chronic or acute myeloid leukemia. Pharmacodynamic analyses suggest that other kinases inhibited by rebastinib, such as TIE2, may be more relevant targets for the clinical development of rebastinib (clinicaltrials.gov Identifier:00827138).

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2.

Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases.

Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells.

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability.

Human ribonuclease with a pendant poly(ethylene glycol) inhibits tumor growth in mice.

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-PEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 µmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ∼5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.

Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the ß-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.

Site-specific PEGylation endows a mammalian ribonuclease with antitumor activity.

Mammalian ribonucleases are emerging as cancer chemotherapeutic agents. Their cationicity engenders cell permeability, and their enzymatic activity destroys the biochemical information encoded by RNA. The pharmacologic potential of ribonucleases is, however, obviated by their high sensitivity to a cytosolic inhibitor protein (RI) and their small size, which limits their residence in serum. We reasoned that site specific conjugation of a poly(ethylene glycol) (PEG) chain could both reduce sensitivity to RI and increase serum half-life. We found that appending a PEG moiety can enable bovine pancreatic ribonuclease (RNase A) to evade RI, depending on the site of conjugation and the length and branching of the chain. Although a pendant PEG moiety decreases antiproliferative activity in vitro, PEGylation discourages renal clearance in vivo and leads to nearly complete tumor growth inhibition in a mouse xenograft model. These data demonstrate that a pendant PEG moiety can be beneficial to the action of proteins that act within the cytosol, and that strategic site-specific PEGylation can endow a mammalian ribonuclease with potent antitumor activity.

Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036.

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

Antitumor activity of ribonuclease multimers created by site-specific covalent tethering.

Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both the site of conjugation and, to a lesser extent, the propinquity of the monomers within the conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth at a lower dose and with less frequent administration than does the monomer. This effect is attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the trimer is ∼300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for the treatment of lung cancer. These data encourage the development of mammalian ribonucleases for the treatment of human cancers.

Evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity.

Onconase (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity--an extraordinary feat considering the modest sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more attractive chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RIribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.

Increasing the potency of a cytotoxin with an arginine graft.

Variants and homologs of bovine pancreatic ribonuclease (RNase A) can exhibit cytotoxic activity. This toxicity relies on cellular internalization of the enzyme. Residues Glu49 and Asp53 form an anionic patch on the surface of RNase A. We find that replacing these two residues with arginine does not affect catalytic activity or affinity for the cytosolic ribonuclease inhibitor (RI) protein. This 'arginine graft' does, however, increase toxicity towards human cancer cells. Appending a nonaarginine domain to this cationic variant results in an additional increase in cytotoxicity, providing one of the most cytotoxic known variants of RNase A. These findings correlate the potency of a ribonuclease with its deliverance of ribonucleolytic activity to the cytosol, and indicate a rational means to enhance the efficacy of ribonucleases and other cytotoxic proteins.

Tuning the pK(a) of fluorescein to optimize binding assays.

The phenolic pKa of fluorescein varies depending on its environment. The fluorescence of the dye varies likewise. Accordingly, a change in fluorescence can report on the association of a fluorescein conjugate to another molecule. Here, we demonstrate how to optimize this process with chemical synthesis. The fluorescence of fluorescein-labeled model protein, bovine pancreatic ribonuclease (RNase A), decreases upon binding to its cognate inhibitor protein (RI). Free and RI-bound fluorescein-RNase A have pKa values of 6.35 and 6.70, respectively, leaving the fluorescein moiety largely unprotonated at physiological pH and thus limiting the sensitivity of the assay. To increase the fluorescein pKa and, hence, the assay sensitivity, we installed an electron-donating alkyl group ortho to each phenol group. 2',7'-Diethylfluorescein (DEF) has spectral properties similar to those of fluorescein but a higher phenolic pKa. Most importantly, free and RI-bound DEF-RNase A have pKa values of 6.68 and 7.29, respectively, resulting in a substantial increase in the sensitivity of the assay. Using DEF-RNase A rather than fluorescein-RNase A in a microplate assay at pH 7.12 increased the Z'-factor from -0.17 to 0.69. We propose that synthetic "tuning" of the pKa of fluorescein and other pH-sensitive fluorophores provides a general means to optimize binding assays.

Disruption of shape-complementarity markers to create cytotoxic variants of ribonuclease A.

Onconase (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI.RNase A complex having a K(d) value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI.RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K(d) value of the pRI.RNase A complex by 20 x 10(6)-fold (to 1.4 microM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 microM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.

Formation, distribution, and elimination of infliximab and anti-infliximab immune complexes in cynomolgus monkeys.

Infliximab (IFX) is a chimeric IgG1 monoclonal antibody specific for human tumor necrosis factor-alpha that is approved in the United States and Europe for the treatment of rheumatoid arthritis (RA) and Crohn's disease (CD). Approximately 10% of RA and CD patients receiving maintenance treatment with IFX will develop antibodies to IFX. The objective of this study was to develop a model to assess the in vivo formation, distribution, and elimination of immune complexes resulting from a low-level immune response in the presence of the excess concentration of a therapeutic antigen. In this model, cynomolgus monkeys were treated with a single intravenous injection of IFX, followed by injection of either radiolabeled, purified monkey anti-IFX IgG antibody (n = 3, test group) or radiolabeled monkey, nonimmune IgG (n = 3, control group). High-performance liquid chromatography analysis of collected sera revealed a rapid formation of immune complexes comprised of IFX and radiolabeled anti-IFX IgG antibody immune complexes. The terminal half-life of the anti-IFX IgG antibody immune complex was approximately 38 h compared with 86 h for the nonimmune antibody. However, the pharmacokinetic profile of IFX, although slightly lower in concentration over time for the test group, was not notably different relative to the control group. There were no macroscopic or microscopic histological findings in either treatment group. These data confirm that immune complexes between IFX and anti-IFX IgG antibodies can form in vivo and that these immune complexes are eliminated more rapidly than nonimmune antibodies in the presence of excess IFX.