Covalent inhibition of pro-apoptotic BAX.

BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.

A small-molecule mimic of a peptide docking motif inhibits the protein kinase PDK1.

There is great interest in developing selective protein kinase inhibitors by targeting allosteric sites, but these sites often involve protein-protein or protein-peptide interfaces that are very challenging to target with small molecules. Here we present a systematic approach to targeting a functionally conserved allosteric site on the protein kinase PDK1 called the PDK1-interacting fragment (PIF)tide-binding site, or PIF pocket. More than two dozen prosurvival and progrowth kinases dock a conserved peptide tail into this binding site, which recruits them to PDK1 to become activated. Using a site-directed chemical screen, we identified and chemically optimized ligand-efficient, selective, and cell-penetrant small molecules (molecular weight ∼ 380 Da) that compete with the peptide docking motif for binding to PDK1. We solved the first high-resolution structure of a peptide docking motif (PIFtide) bound to PDK1 and mapped binding energy hot spots using mutational analysis. We then solved structures of PDK1 bound to the allosteric small molecules, which revealed a binding mode that remarkably mimics three of five hot-spot residues in PIFtide. These allosteric small molecules are substrate-selective PDK1 inhibitors when used as single agents, but when combined with an ATP-competitive inhibitor, they completely suppress the activation of the downstream kinases. This work provides a promising new scaffold for the development of high-affinity PIF pocket ligands, which may be used to enhance the anticancer activity of existing PDK1 inhibitors. Moreover, our results provide further impetus for exploring the helix αC patches of other protein kinases as potential therapeutic targets even though they involve protein-protein interfaces.

Novel Corrector for Variants of SLC6A8: A Therapeutic Opportunity for Creatine Transporter Deficiency.

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD. We leveraged a novel chemoproteomic technology for ligand discovery, reactive affinity probe interaction discovery, to identify small-molecule fragments with photoaffinity handles that bind to SLC6A8 in a cellular environment. We synthesized a library of irreversible covalent analogs of these molecules to characterize in functional assays, which revealed molecules that could promote the trafficking of mutant SLC6A8 variants to the cell surface. Further medicinal chemistry was able to identify reversible drug-like small molecules that both promoted trafficking of the transporter and also rescued creatine uptake. When profiled across the 27 most prevalent SLC6A8 missense variants, we found that 10-20% of patient mutations were amenable to correction by our molecules. These results were verified in an endogenous setting using the CRISPR knock-in of selected missense alleles. We established in vivo proof-of-mechanism for correctors in a novel CTD mouse model with the P544L patient-defined variant knocked in to the SLC6A8 locus, where treatment with our orally bioavailable and brain penetrant tool corrector increased brain creatine levels in heterozygous female mice, validating correctors as a potential therapeutic approach for CTD.

Redox priming promotes Aurora A activation during mitosis.

Cell cycle-dependent redox changes can mediate transient covalent modifications of cysteine thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle phases suggests that redox modifications could play prominent roles in regulating mitotic processes. The Aurora family of kinases and their downstream targets are key components of the cellular machinery that ensures the proper execution of mitosis and the accurate segregation of chromosomes to daughter cells. In this study, x-ray crystal structures of the Aurora A kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can allosterically regulate kinase activity and oligomerization state. We showed in both Xenopus laevis egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain. These findings reveal a potential mechanistic link between Aurora A activation and changes in the intracellular redox state during mitosis and provide insights into how novel small-molecule inhibitors may be developed to target specific subpopulations of Aurora A.

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

CryptoSite: Expanding the Druggable Proteome by Characterization and Prediction of Cryptic Binding Sites.

Many proteins have small-molecule binding pockets that are not easily detectable in the ligand-free structures. These cryptic sites require a conformational change to become apparent; a cryptic site can therefore be defined as a site that forms a pocket in a holo structure, but not in the apo structure. Because many proteins appear to lack druggable pockets, understanding and accurately identifying cryptic sites could expand the set of drug targets. Previously, cryptic sites were identified experimentally by fragment-based ligand discovery and computationally by long molecular dynamics simulations and fragment docking. Here, we begin by constructing a set of structurally defined apo-holo pairs with cryptic sites. Next, we comprehensively characterize the cryptic sites in terms of their sequence, structure, and dynamics attributes. We find that cryptic sites tend to be as conserved in evolution as traditional binding pockets but are less hydrophobic and more flexible. Relying on this characterization, we use machine learning to predict cryptic sites with relatively high accuracy (for our benchmark, the true positive and false positive rates are 73% and 29%, respectively). We then predict cryptic sites in the entire structurally characterized human proteome (11,201 structures, covering 23% of all residues in the proteome). CryptoSite increases the size of the potentially "druggable" human proteome from ~40% to ~78% of disease-associated proteins. Finally, to demonstrate the utility of our approach in practice, we experimentally validate a cryptic site in protein tyrosine phosphatase 1B using a covalent ligand and NMR spectroscopy. The CryptoSite Web server is available at http://salilab.org/cryptosite.

Small-Molecule Allosteric Modulators of the Protein Kinase PDK1 from Structure-Based Docking.

Finding small molecules that target allosteric sites remains a grand challenge for ligand discovery. In the protein kinase field, only a handful of highly selective allosteric modulators have been found. Thus, more general methods are needed to discover allosteric modulators for additional kinases. Here, we use virtual screening against an ensemble of both crystal structures and comparative models to identify ligands for an allosteric peptide-binding site on the protein kinase PDK1 (the PIF pocket). We optimized these ligands through an analog-by-catalog search that yielded compound 4, which binds to PDK1 with 8 µM affinity. We confirmed the docking poses by determining a crystal structure of PDK1 in complex with 4. Because the PIF pocket appears to be a recurring structural feature of the kinase fold, known generally as the helix αC patch, this approach may enable the discovery of allosteric modulators for other kinases.

FP Tethering: a screening technique to rapidly identify compounds that disrupt protein-protein interactions.

Tethering is a screening technique for discovering small-molecule fragments that bind to pre-determined sites via formation of a disulphide bond. Tethering screens traditionally rely upon mass spectrometry to detect disulphide bind formation, which requires a time-consuming liquid chromatography step. Here we show that Tethering can be performed rapidly and inexpensively using a homogenous fluorescence polarization (FP) assay that detects displacement of a peptide ligand from the protein target as an indirect readout of disulphide formation. We apply this method, termed FP Tethering, to identify fragments that disrupt the protein-protein interaction between the KIX domain of the transcriptional coactivator CBP and the transcriptional activator peptide pKID.