GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model.

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.

A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.

Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

Increased hemoglobin affinity for oxygen with GBT1118 improves hypoxia tolerance in sickle cell mice.

Therapeutic agents that increase the Hb affinity for oxygen (O2) could, in theory, lead to decreased O2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O2, was used to assess the impact of increasing Hb affinity for O2 on brain tissue oxygenation, blood pressure, heart rate, O2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O2, reducing the Po2 for 50% HbO2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O2 delivery and extraction. Chronically increasing Hb affinity for O2 with GBT1118 preserved cortical O2 tension during normoxia, improved cortical O2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD.NEW & NOTEWORTHY Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4(CRBN). These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates.

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.

Cereblon modulators: Low molecular weight inducers of protein degradation.

Targeted protein degradation has become an exciting new paradigm in drug discovery with the potential to target new protein families for therapeutic intervention. In 2010, Hiroshi Handa and colleagues discovered that the drug thalidomide binds to the protein cereblon, a component of the CRL4CRBN E3 ubiquitin ligase. In contrast to the heterobifunctional small molecule degraders reported in the literature, thalidomide is of very low molecular weight (∼258Da) with molecular properties (solubility, metabolic stability, permeability etc) that readily support pharmaceutical dosing. It was subsequently shown that thalidomide and the analogues lenalidomide and pomalidomide are able to degrade the transcription factors Ikaros and Aiolos. CK1α and GSPT1 were subsequently identified as substrates for specific ligands, indicating that this molecular class could be tuned for selective protein degradation. Structural studies showed that the thalidomide analogues bind to a shallow hydrophobic pocket on the surface of cereblon, and scaffold a protein-protein interaction with target proteins. Target proteins do not need any affinity for the cereblon modulators, and as such undruggable, or even unligandable, proteins can be targeted for degradation. A similar mechanism of action was subsequently identified for the clinical molecule indisulam, indicating that low molecular weight degraders are not unique to cereblon. The groundbreaking work on cereblon represents a case study for the discovery and characterization of low molecular weight protein degraders for other ligases.

Pomalidomide in combination with dexamethasone results in synergistic anti-tumour responses in pre-clinical models of lenalidomide-resistant multiple myeloma.

Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Discovery of CC-930, an orally active anti-fibrotic JNK inhibitor.

In this Letter we describe the discovery of potent, selective, and orally active aminopurine JNK inhibitors. Improving the physico-chemical properties as well as increasing the potency and selectivity of a subseries with rat plasma exposure, led to the identification of four structurally diverse inhibitors. Differentiation based on PK profiles in multiple species as well as activity in a chronic efficacy model led to the identification of 1 (CC-930) as a development candidate, which is currently in Phase II clinical trial for IPF.

Aminopurine based JNK inhibitors for the prevention of ischemia reperfusion injury.

In this Letter we describe the optimization of an aminopurine lead (1) with modest potency and poor overall kinase selectivity which led to the identification of a series of potent, selective JNK inhibitors. Improvement in kinase selectivity was enabled by introduction of an aliphatic side chain at the C-2 position. CC-359 (2) was selected as a potential clinical candidate for diseases manifested by ischemia reperfusion injury.

CC-90009: A Cereblon E3 Ligase Modulating Drug That Promotes Selective Degradation of GSPT1 for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is marked by significant unmet clinical need due to both poor survival and high relapse rates where long-term disease control for most patients with relapsed or refractory AML remain dismal. Inspired to bring novel therapeutic options to these patients, we envisioned protein degradation as a potential therapeutic approach for the treatment of AML. Following this course, we discovered and pioneered a novel mechanism of action which culminated in the discovery of CC-90009. CC-90009 represents a novel protein degrader and the first cereblon E3 ligase modulating drug to enter clinical development that specifically targets GSPT1 (G1 to S phase transition 1) for proteasomal degradation. This manuscript briefly summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and efficacy data for CC-90009, which is currently in phase 1 clinical development.

Discovery of the Selective Protein Kinase C-θ Kinase Inhibitor, CC-90005.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos.

The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modulator in clinical development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compound 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degradation of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compound 6 reveals that the increase in potency correlates with increased contacts between compound 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degradation.

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.

Cellular transformation of the investigational new anticancer drug NB1011, a phosphoramidate of 5-(2-bromovinyl)-2'-deoxyuridine, results in modification of cellular proteins not DNA.

NB1011 [E-5-(2-bromovinyl)-2'-deoxyuridine-5'-(L-methylalaninyl)-phenylphosphoramidate], a phosphoramidate prodrug of E-5-(2-bromovinyl)-2'-deoxyuridine-5'-monophosphate (BVdUMP), is an investigational new anticancer drug. NB1011 targets thymidylate synthase (TS), which catalyzes the transformation of BVdUMP into cytotoxic reaction products. Due to the elevated levels of TS expression in tumor cells compared to normal cells, these cytotoxic products are preferentially generated inside tumor cells, and, as expected, NB1011 is more toxic to cells with higher levels of TS expression. Therefore, NB1011 therapy should kill tumor cells without severely damaging normal cells. Radiolabeled NB1011 was used to determine the intracellular fate of NB1011 reaction products and, possibly, the mechanism of action of this investigational new drug. We found significant incorporation of the radiolabel into cellular macromolecules. In contrast to our expectations that NB1011 product(s) would be incorporated into DNA, we discovered that cellular proteins were the labeled macromolecular fraction. Herein, we report that the intracellular transformation of NB1011 involves formation of the corresponding monophosphate, TS-dependent transformation into highly reactive intermediates, and subsequent incorporation into cellular proteins. TS itself appears to escape irreversible inactivation. Our data suggest that protein modification not DNA incorporation accounts for the therapeutic effect of NB1011. The proposed mechanism is rather unexpected for a nucleotide analogue and could lead to the discovery of new cellular protein targets for future drug design.

Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs.

The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.

Substrate recognition and ubiquitination of SCFSkp2/Cks1 ubiquitin-protein isopeptide ligase.

p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.

pH Dependence of inhibitors targeting the occluding loop of cathepsin B.

Potent and selective cathepsin B inhibitors have previously been synthesized based upon the natural product cysteine protease inhibitor E-64. X-ray crystal data indicates that these compounds interact through their free carboxylate with the positively charged histidine residues located on the prime-side of the active site within the occluding loop of cathepsin B. Herein, we examine the pH dependence of two prime-side-binding compounds. In each case there is a dramatic decrease in k(inact)/K(I) as the pH is raised from 4 to 7.8 corresponding to a single ionization of pK(a) 4.4. These results suggest that targeting of the occluding loop of cathepsin B may be a poor inhibitor design strategy if the enzyme environment has a pH greater than 5.5. However, this type of inhibitor may be a useful tool to help elucidate the role and the environment of cathepsin B in invading tumors.