Selective Wee1 degradation by PROTAC degraders recruiting VHL and CRBN E3 ubiquitin ligases.

The Ser/Thr protein kinase Wee1 plays a regulatory role at the G2/M checkpoint by phosphorylating CDK1 when DNA is damaged to allow time for DNA to repair, disruption of which is a key approach to sensitise cancer cells to DNA-damaging therapies. The main selective inhibitor for Wee1 undergoing development in clinical trials, AZD1775, however, has been shown to have off target effects towards other protein kinases with similar potency. Here we describe the synthesis and assessment of a series of Wee1-degrading PROTACs using AZD1775 linked to either the VHL ligand VH032 or to the CRBN ligand pomalidomide using different types and lengths of linkers. The conversion of AZD1775 into a PROTAC induces selective Wee1 degradation for compounds of both series depending on the nature of the linker.

3-Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation.

Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e., fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-l-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy, and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3 R,4 S)-F-Hyp over the corresponding (3 S,4 S) epimer by VHL. We show that (3 R,4 S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia-inducible factor 1 alpha (HIF-1α) substrate peptides and peptidomimetic ligands that form part of PROTAC (proteolysis targeting chimera) conjugates for targeted protein degradation. Despite a weakened affinity, Hyp substitution with (3 S,4 S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organic chemists, chemical biologists, and drug discoverers alike.

Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298).

The von Hippel-Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein-protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure-activity relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways and new VHL ligands for next-generation PROTACs.

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting.

Structural basis of PROTAC cooperative recognition for selective protein degradation.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition.

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

Serendipitous SAD Solution for DMSO-Soaked SOCS2-ElonginC-ElonginB Crystals Using Covalently Incorporated Dimethylarsenic: Insights into Substrate Receptor Conformational Flexibility in Cullin RING Ligases.

Suppressor of cytokine signalling 2 (SOCS2) is the substrate-binding component of a Cullin-RING E3 ubiquitin ligase (CRL) complex that targets phosphorylated hormone receptors for degradation by the ubiquitin-proteasome system. As a key regulator of the transcriptional response to growth signals, SOCS2 and its protein complex partners are potential targets for small molecule development. We found that crystals of SOCS2 in complex with its adaptor proteins, Elongin C and Elongin B, underwent a change in crystallographic parameters when treated with dimethyl sulfoxide during soaking experiments. To solve the phase problem for the new crystal form we identified the presence of arsenic atoms in the crystals, a result of covalent modification of cysteines by cacodylate, and successfully extracted anomalous signal from these atoms for experimental phasing. The resulting structure provides a means for solving future structures where the crystals must be treated with DMSO for ligand soaking approaches. Additionally, the conformational changes induced in this structure reveal flexibility within SOCS2 that match those postulated by previous molecular dynamics simulations. This conformational flexibility illustrates how SOCS2 can orient its substrates for successful ubiquitination by other elements of the CRL complex.

Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes.

Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine-contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2-C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates. The kinetic parameters of DmrA with 4-bromobutyronitrile as a substrate were Km = 1.9 ± 0.2 mM, kcat = 3.1 ± 0.2 s(-1) . DmrB showed the highest activity against 1-bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side-chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. We show that M. rhodesiae JS60 can utilise 1-iodopropane, 1-iodobutane and 1-bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.

Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities.

E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities.

A structural basis for the regulation of the LIM-homeodomain protein islet 1 (Isl1) by intra- and intermolecular interactions.

Islet 1 (Isl1) is a transcription factor of the LIM-homeodomain (LIM-HD) protein family and is essential for many developmental processes. LIM-HD proteins all contain two protein-interacting LIM domains, a DNA-binding homeodomain (HD), and a C-terminal region. In Isl1, the C-terminal region also contains the LIM homeobox 3 (Lhx3)-binding domain (LBD), which interacts with the LIM domains of Lhx3. The LIM domains of Isl1 have been implicated in inhibition of DNA binding potentially through an intramolecular interaction with or close to the HD. Here we investigate the LBD as a candidate intramolecular interaction domain. Competitive yeast-two hybrid experiments indicate that the LIM domains and LBD from Isl1 can interact with apparently low affinity, consistent with no detection of an intermolecular interaction in the same system. Nuclear magnetic resonance studies show that the interaction is specific, whereas substitution of the LBD with peptides of the same amino acid composition but different sequence is not specific. We solved the crystal structure of a similar but higher affinity complex between the LIM domains of Isl1 and the LIM interaction domain from the LIM-HD cofactor protein LIM domain-binding protein 1 (Ldb1) and used these coordinates to generate a homology model of the intramolecular interaction that indicates poorer complementarity for the weak intramolecular interaction. The intramolecular interaction in Isl1 may provide protection against aggregation, minimize unproductive DNA binding, and facilitate cofactor exchange within the cell.

Crystallization and diffraction of an Isl1-Ldb1 complex.

A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Isl1 tethered to a peptide region of Ldb1 has been engineered, purified and crystallized. The orthorhombic crystals belonged to space group P222(1), with unit-cell parameters a=57.2, b=56.7, c=179.8 Å, and diffracted to 3.10 Šresolution.

Solution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions.

Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.

Structural basis for partial redundancy in a class of transcription factors, the LIM homeodomain proteins, in neural cell type specification.

Combinations of LIM homeodomain proteins form a transcriptional "LIM code" to direct the specification of neural cell types. Two paralogous pairs of LIM homeodomain proteins, LIM homeobox protein 3/4 (Lhx3/Lhx4) and Islet-1/2 (Isl1/Isl2), are expressed in developing ventral motor neurons. Lhx3 and Isl1 interact within a well characterized transcriptional complex that triggers motor neuron development, but it was not known whether Lhx4 and Isl2 could participate in equivalent complexes. We have identified an Lhx3-binding domain (LBD) in Isl2 based on sequence homology with the Isl1(LBD) and show that both Isl2(LBD) and Isl1(LBD) can bind each of Lhx3 and Lhx4. X-ray crystal- and small-angle x-ray scattering-derived solution structures of an Lhx4·Isl2 complex exhibit many similarities with that of Lhx3·Isl1; however, structural differences supported by mutagenic studies reveal differences in the mechanisms of binding. Differences in binding have implications for the mode of exchange of protein partners in transcriptional complexes and indicate a divergence in functions of Lhx3/4 and Isl1/2. The formation of weaker Lhx·Isl complexes would likely be masked by the availability of the other Lhx·Isl complexes in postmitotic motor neurons.

Crystallization and diffraction of an Lhx4-Isl2 complex.

A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. The monoclinic crystals belonged to space group P2(1), with unit-cell parameters a = 46.8, b = 88.7, c = 49.9 A, beta = 111.9 degrees, and diffracted to 2.16 A resolution.