Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design.

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases.

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.

Performance of a docking/molecular dynamics protocol for virtual screening of nutlin-class inhibitors of Mdmx.

A virtual screening protocol involving docking and molecular dynamics has been tested against the results of fluorescence polarization assays testing the potency of a series of compounds of the nutlin class for inhibition of the interaction between p53 and Mdmx, an interaction identified as a driver of certain cancers. The protocol uses a standard docking method (AutoDock) with a cutoff based on the AutoDock score (ADscore), followed by molecular dynamics simulation with a cutoff based on root-mean-square-deviation (RMSD) from the docked pose. An analysis of the experimental and computational results shows modest performance of ADscore alone, but dramatically improved performance when RMSD is also used.

Monitoring Ligand-Induced Protein Ordering in Drug Discovery.

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: A platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition. J. Org. Chem. 79, 6913-6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity (Kd value of 150nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly (Kd values from 600nM to 12µM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21(Cip1) expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure-activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation.

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells.

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: a platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition.

The finding by scientists at Hoffmann-La Roche that cis-imidazolines could disrupt the protein-protein interaction between p53 and MDM2, thereby inducing apoptosis in cancer cells, raised considerable interest in this scaffold over the past decade. Initial routes to these small molecules (i.e., Nutlin-3) provided only the racemic form, with enantiomers being enriched by chromatographic separation using high-pressure liquid chromatography (HPLC) and a chiral stationary phase. Reported here is the first application of an enantioselective aza-Henry approach to nonsymmetric cis-stilbene diamines and cis-imidazolines. Two novel mono(amidine) organocatalysts (MAM) were discovered to provide high levels of enantioselection (>95% ee) across a broad range of substrate combinations. Furthermore, the versatility of the aza-Henry strategy for preparing nonsymmetric cis-imidazolines is illustrated by a comparison of the roles of aryl nitromethane and aryl aldimine in the key step, which revealed unique substrate electronic effects providing direction for aza-Henry substrate-catalyst matching. This method was used to prepare highly substituted cis-4,5-diaryl imidazolines that project unique aromatic rings, and these were evaluated for MDM2-p53 inhibition in a fluorescence polarization assay. The diversification of access to cis-stilbene diamine-derived imidazolines provided by this platform should streamline their further development as chemical tools for disrupting protein-protein interactions.

Slow amyloid nucleation via α-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments.

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in ß-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ ß-sheet aggregates. These final amyloid-like aggregates not only feature the expected high ß-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.

A structure-activity relationship study of small-molecule inhibitors of GLI1-mediated transcription.

We have previously reported ketoprofen amide compounds as inhibitors of GLI1-mediated transcription, an essential down-stream element of the Hedgehog (Hh) pathway. These compounds inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1. Here we have designed new derivatives of these compounds aiming to explore the structure-activation relationship (SAR). By replacing the ketone carbonyl group of the ketoprofen moiety with an ether, amide, sulfonamide, or sulfone, we found several new compounds that are equipotent to the ketoprofen amide compounds. Among them, sulfone 30 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1.

PqsD is responsible for the synthesis of 2,4-dihydroxyquinoline, an extracellular metabolite produced by Pseudomonas aeruginosa.

2,4-Dihydroxyquinoline (DHQ) is an abundant extracellular metabolite of the opportunistic pathogen Pseudomonas aeruginosa that is secreted into growth medium in stationary phase to concentrations comparable with those of the Pseudomonas quinolone signal. Using a combination of biochemical and genetic approaches, we show that PqsD, a condensing enzyme in the pqs operon that is essential for Pseudomonas quinolone signal synthesis, accounts for DHQ formation in vivo. First, the anthraniloyl moiety is transferred to the active-site Cys of PqsD to form an anthraniloyl-PqsD intermediate, which then condenses with either malonyl-CoA or malonyl-acyl carrier protein to produce 3-(2-aminophenyl)-3-oxopropanoyl-CoA. This short-lived intermediate undergoes an intramolecular rearrangement to form DHQ. DHQ was produced by Escherichia coli coexpressing PqsA and PqsD, illustrating that these two proteins are the only factors necessary for DHQ synthesis. Thus, PqsD is responsible for the production of DHQ in P. aeruginosa.

Rational design of the first small-molecule antagonists of NHERF1/EBP50 PDZ domains.

This report describes the first small-molecule antagonists that specifically target the ligand-binding pocket of PDZ domains of NHERF1 multi-functional adaptor protein. Comparison of the peptide sequence homology between the native ligand of NHERF1 PDZ domains and an indole-based non-peptide chemical scaffold allowed the design of a small-molecule antagonist of NHERF1 PDZ domains.

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.

The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements.

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.

Assignment of the four disulfides in the N-terminal somatomedin B domain of native vitronectin isolated from human plasma.

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys(5):Cys(9), Cys(19):Cys(31), Cys(21):Cys(32), and Cys(25):Cys(39). This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys(19): Cys(31) and Cys(21):Cys(32) disulfides. A small positively charged loop is created at the N terminus by the Cys(5): Cys(9) cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys(25) and Cys(39) similar to cystine-stabilized alpha-helical structures commonly observed in cystine knots. This alpha-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor.