Intrinsically Disordered Regions in the Transcription Factor MYC:MAX Modulate DNA Binding via Intramolecular Interactions.

The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large part an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promoters containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins in turn regulates MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We used nuclear magnetic resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA-binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e., as intramolecular interactions. These findings are supported by surface plasmon resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights into how bHLH-LZ TFs are regulated by intramolecular interactions, which open up new possibilities for drug discovery.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Discovery of New Binders for DCAF1, an Emerging Ligase Target in the Targeted Protein Degradation Field.

In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected on the basis of internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration.

The Disordered MAX N-terminus Modulates DNA Binding of the Transcription Factor MYC:MAX.

The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

Optimization of the In Vivo Potency of Pyrazolopyrimidine MALT1 Protease Inhibitors by Reducing Metabolism and Increasing Potency in Whole Blood.

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.

Structure-Based Design and Preclinical Characterization of Selective and Orally Bioavailable Factor XIa Inhibitors: Demonstrating the Power of an Integrated S1 Protease Family Approach.

The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

An allosteric MALT1 inhibitor is a molecular corrector rescuing function in an immunodeficient patient.

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.

N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of MALT1 proteolytic activity.

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.

The T-cell fingerprint of MALT1 paracaspase revealed by selective inhibition.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.

Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

Azaindoles as Zinc-Binding Small-Molecule Inhibitors of the JAMM Protease CSN5.

CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.

Targeted inhibition of the COP9 signalosome for treatment of cancer.

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin-proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.

Crystal structure of the human COP9 signalosome.

Ubiquitination is a crucial cellular signalling process, and is controlled on multiple levels. Cullin-RING E3 ubiquitin ligases (CRLs) are regulated by the eight-subunit COP9 signalosome (CSN). CSN inactivates CRLs by removing their covalently attached activator, NEDD8. NEDD8 cleavage by CSN is catalysed by CSN5, a Zn(2+)-dependent isopeptidase that is inactive in isolation. Here we present the crystal structure of the entire ∼350-kDa human CSN holoenzyme at 3.8 Å resolution, detailing the molecular architecture of the complex. CSN has two organizational centres: a horseshoe-shaped ring created by its six proteasome lid-CSN-initiation factor 3 (PCI) domain proteins, and a large bundle formed by the carboxy-terminal α-helices of every subunit. CSN5 and its dimerization partner, CSN6, are intricately embedded at the core of the helical bundle. In the substrate-free holoenzyme, CSN5 is autoinhibited, which precludes access to the active site. We find that neddylated CRL binding to CSN is sensed by CSN4, and communicated to CSN5 with the assistance of CSN6, resulting in activation of the deneddylase.

Phosphorylation meets proteolysis.

Phosphorylation is a reversible post-translational modification that regulates many proteins and enzymes, including proteases, as shown by two recent publications. Huang and colleagues and Velázquez-Delgado and Hardy (this issue of Structure) describe how phosphorylation activates the protease activity of the deubiquitinating enzyme DUBA and how it inhibits caspase-6, respectively.

Structural determinants of MALT1 protease activity.

The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space.

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1-P1') but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors--namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.

The crystal structure of caspase-6, a selective effector of axonal degeneration.

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.