Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors.

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.

Quantitative Chemical Proteomic Profiling of the in Vivo Targets of Reactive Drug Metabolites.

Idiosyncratic liver toxicity represents an important problem in drug research and pharmacotherapy. Reactive drug metabolites that modify proteins are thought to be a principal factor in drug-induced liver injury. Here, we describe a quantitative chemical proteomic method to identify the targets of reactive drug metabolites in vivo. Treating mice with clickable analogues of four representative hepatotoxic drugs, we demonstrate extensive covalent binding that is confined primarily to the liver. Each drug exhibited a distinct target profile that, in certain cases, showed strong enrichment for specific metabolic pathways (e.g., lipid/sterol pathways for troglitazone). Site-specific proteomics revealed that acetaminophen reacts with high stoichiometry with several conserved, functional (seleno)cysteine residues throughout the liver proteome. Our findings thus provide an advanced experimental framework to characterize the proteomic reactivity of drug metabolites in vivo, revealing target profiles that may help to explain mechanisms and identify risk factors for drug-induced liver injury.

Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors.

Electrophilic small molecules are an important class of chemical probes and drugs that produce biological effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug dimethyl fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small molecules as chemical probes and therapeutics. Here, we describe an approach to confer kinetic selectivity to electrophilic drugs. We show that an analogue of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymatic metabolism on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochemical pathways with greater precision.

Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein.

UNLABELLED: Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCE: Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS.

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.

A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative.

The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.

Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis.

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-ß (DAGLß), a principal biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (2-substituted)-piperidyl-1,2,3-TUs that selectively inactivate DAGLß in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts. We also describe an expanded repertoire of DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,2,3-TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic pathways.

Discovery and optimization of piperidyl-1,2,3-triazole ureas as potent, selective, and in vivo-active inhibitors of α/ß-hydrolase domain containing 6 (ABHD6).

α/ß-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (<5 nM) and, at equivalent doses in mice (1 mg kg(-1)), act as systemic and peripherally restricted ABHD6 inhibitors, respectively. We also describe an orally bioavailable ABHD6 inhibitor, KT185, that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models.

NMR-assisted computational studies of peptidomimetic inhibitors bound in the hydrophobic pocket of HIV-1 glycoprotein 41.

Due to the inherently flexible nature of a protein-protein interaction surface, it is difficult both to inhibit the association with a small molecule, and to predict how it might bind to the surface. In this study, we have examined small molecules that mediate the interaction between a WWI motif on the C-helix of HIV-1 glycoprotein-41 (gp41) and a deep hydrophobic pocket contained in the interior N-helical trimer. Association between these two components of gp41 leads to virus-cell and cell-cell fusion, which could be abrogated in the presence of an inhibitor that binds tightly in the pocket. We have studied a comprehensive combinatorial library of α-helical peptidomimetics, and found that compounds with strongly hydrophobic side chains had the highest affinity. Computational docking studies produced multiple possible binding modes due to the flexibility of both the binding site and the peptidomimetic compounds. We applied a transferred paramagnetic relaxation enhancement experiment to two selected members of the library, and showed that addition of a few experimental constraints enabled definitive identification of unique binding poses. Computational docking results were extremely sensitive to side chain conformations, and slight variations could preclude observation of the experimentally validated poses. Different receptor structures were required for docking simulations to sample the correct pose for the two compounds. The study demonstrated the sensitivity of predicted poses to receptor structure and indicated the importance of experimental verification when docking to a malleable protein-protein interaction surface.

Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2).

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.

Comprehensive peptidomimetic libraries targeting protein-protein interactions.

Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, ß-turn, and ß-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member ß-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or ß-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or ß-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (ß-turn mimetic library) led to the discovery of library members expected to mimic the known endogenous ligands. These efforts led to the discovery of high-affinity α-helix mimetics (K(i) = 0.7 µM) against HIV-1 gp41 as well as high-affinity and selective ß-turn mimetics (K(i) = 80 nM) against the κ-opioid receptor. The results suggest that the use of such comprehensive libraries of peptide secondary structure mimetics, built around effective molecular scaffolds, constitutes a powerful method of interrogating PPIs. These structures provide small molecule modulators of PPI networks for therapeutic target validation, lead compound discovery, and the identification of modulators of biological processes for further study.

Discovery of HIV fusion inhibitors targeting gp41 using a comprehensive α-helix mimetic library.

The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic pocket recognizing an intramolecular CHR α-helix provided a detailed depiction of structural features required for binding and led to the discovery of small molecule inhibitors (K(i) 0.6-1.3 µM) that not only match or exceed the potency of those disclosed over the past decade, but that also exhibit effective activity in a cell-cell fusion assay (IC(50) 5-8 µM).

Design, synthesis, and validation of a ß-turn mimetic library targeting protein-protein and peptide-receptor interactions.

The design and synthesis of a ß-turn mimetic library as a key component of a small-molecule library targeting the major recognition motifs involved in protein-protein interactions is described. Analysis of a geometric characterization of 10,245 ß-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a ß-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities (K(i) = 390 and 23 nM, respectively) for the κ-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring ß-turn. Unique to the design and because of the C(2) symmetry of the template, a typical 20 × 20 × 20-mix (8000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid-liquid or liquid-solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as K(i) = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-Pro-Trp/Phe-Phe-NH(2) ß-turn is important for MOR binding but may not be important for KOR (accommodated, but not preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a chlorine substituent, further enhancing KOR affinity.

Small molecule peptidomimetic inhibitors of importin α/ß mediated nuclear transport.

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the ß-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/ß pathway. By analyzing peptidomimetic libraries, we identified ß-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/ß but not by transportin. Structure-activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors.

Identification of broad-based HIV-1 protease inhibitors from combinatorial libraries.

Clinically approved inhibitors of the HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature infectious viral particle. Advances in chemical synthesis have led to the development of new high-diversity chemical libraries using rapid in-solution syntheses. These libraries have been shown previously to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44000 compounds from such a library to identify inhibitors of the HIV-1 protease. One compound was identified that inhibits wild-type protease, as well as a drug-resistant protease with six mutations. Moreover, analysis of this compound suggests an allosteric non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy.

Small molecule inhibitors of Myc/Max dimerization and Myc-induced cell transformation.

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced cell transformation are described providing mycmycin-1 (3) and mycmycin-2 (4).