Landon Whitby - Academia.edu (original) (raw)

Papers by Landon Whitby

Proceedings of the National Academy of Sciences of the United States of America, 2016

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and ot... more 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 conta...

Bioorganic & Medicinal Chemistry Letters, 2009

The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell... more The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the more potent (-)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer. The absolute configuration of (-)-3 was established by asymmetric synthesis of the active inhibitor (-)-(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of the prototypical inhibitors.

Biochemical and Biophysical Research Communications, 2003

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase t... more The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the a subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Journal of the American Chemical Society, Jan 14, 2016

Electrophilic small molecules are an important class of chemical probes and drugs that produce bi... more 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 t...

Journal of Virology, 2016

Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Afri... more 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. 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.

Collection Symposium Series, 2005

Bioorganic Medicinal Chemistry Letters, Jan 11, 2009

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced ... more 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).

Nature Chemical Biology, 2014

Kinases are principal components of signal transduction pathways and the focus of intense basic a... more 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.

Nucleic Acids Research, 2006

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Ac... more The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3-to 5-fold by siRNA duplexes with 19 bp and 2 nt 3 0 -overhangs, whereas the maximum activation observed for poly(I)poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N 2 -benzyl-2 0 -deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Journal of the American Chemical Society, 2011

The design and synthesis of a β-turn mimetic library as a key component of a small-molecule libra... more The design and synthesis of a β-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 β-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 β-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 β-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) β-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.

Journal of the American Chemical Society, 2009

The design and solution-phase synthesis of an α-helix mimetic library as an integral component of... more The design and solution-phase synthesis of an α-helix mimetic library as an integral component of a small molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate α-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 × 20 × 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i+4, i+7 amino acid side chains of an α-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquidliquid extractions for purification on a scale that insures its long term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead α-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an α-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

Journal of Medicinal Chemistry, 2013

α/β-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the... more α/β-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.

Journal of Medicinal Chemistry, 2013

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irrevers... more 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-β (DAGLβ), 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 DAGLβ 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.

Bioorganic & Medicinal Chemistry Letters, 2013

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotei... more 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.

Bioorganic & Medicinal Chemistry Letters, 2009

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced ... more 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).

Bioorganic & Medicinal Chemistry Letters, 2012

The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic po... more 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).

Bioorganic & Medicinal Chemistry, 2010

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Trans... more 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 β-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/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β 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.

Biochemical Journal, 2010

Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particul... more Clinically approved inhibitors of 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 chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 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 antiretroviral therapy. screened the combinatorial chemical library for HIV-1 protease inhibitory activity. Ying C. Lin and Jeremiah Savage produced the protease for the biochemical assays and protease screening. Sukwon Hong, Wei Jin, and Landon R. Whitby synthesized, determined the composition and purity of the library as well as deconvoluted the library. Michael J. Giffin and Max W. Chang designed and performed all biochemical analyses on the selected protease inhibitors. Max W. Chang performed all docking studies. John H. Elder, Dale L. Boger, and Bruce E. Torbett were involved in the design and interpretation of the results. Michael J. Giffin, Max W. Chang, and Bruce E. Torbett were primarily involved in writing the manuscript. Bruce E.

Proceedings of the National Academy of Sciences of the United States of America, 2016

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and ot... more 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 conta...

Bioorganic & Medicinal Chemistry Letters, 2009

The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell... more The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the more potent (-)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer. The absolute configuration of (-)-3 was established by asymmetric synthesis of the active inhibitor (-)-(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of the prototypical inhibitors.

Biochemical and Biophysical Research Communications, 2003

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase t... more The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the a subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Journal of the American Chemical Society, Jan 14, 2016

Electrophilic small molecules are an important class of chemical probes and drugs that produce bi... more 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 t...

Journal of Virology, 2016

Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Afri... more 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. 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.

Collection Symposium Series, 2005

Bioorganic Medicinal Chemistry Letters, Jan 11, 2009

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced ... more 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).

Nature Chemical Biology, 2014

Kinases are principal components of signal transduction pathways and the focus of intense basic a... more 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.

Nucleic Acids Research, 2006

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Ac... more The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3-to 5-fold by siRNA duplexes with 19 bp and 2 nt 3 0 -overhangs, whereas the maximum activation observed for poly(I)poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N 2 -benzyl-2 0 -deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Journal of the American Chemical Society, 2011

The design and synthesis of a β-turn mimetic library as a key component of a small-molecule libra... more The design and synthesis of a β-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 β-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 β-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 β-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) β-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.

Journal of the American Chemical Society, 2009

The design and solution-phase synthesis of an α-helix mimetic library as an integral component of... more The design and solution-phase synthesis of an α-helix mimetic library as an integral component of a small molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate α-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 × 20 × 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i+4, i+7 amino acid side chains of an α-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquidliquid extractions for purification on a scale that insures its long term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead α-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an α-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

Journal of Medicinal Chemistry, 2013

α/β-Hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the... more α/β-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.

Journal of Medicinal Chemistry, 2013

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irrevers... more 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-β (DAGLβ), 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 DAGLβ 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.

Bioorganic & Medicinal Chemistry Letters, 2013

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotei... more 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.

Bioorganic & Medicinal Chemistry Letters, 2009

The preparation and evaluation of a series of inhibitors of Myc/Max dimerization and Myc-induced ... more 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).

Bioorganic & Medicinal Chemistry Letters, 2012

The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic po... more 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).

Bioorganic & Medicinal Chemistry, 2010

Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Trans... more 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 β-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/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β 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.

Biochemical Journal, 2010

Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particul... more Clinically approved inhibitors of 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 chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 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 antiretroviral therapy. screened the combinatorial chemical library for HIV-1 protease inhibitory activity. Ying C. Lin and Jeremiah Savage produced the protease for the biochemical assays and protease screening. Sukwon Hong, Wei Jin, and Landon R. Whitby synthesized, determined the composition and purity of the library as well as deconvoluted the library. Michael J. Giffin and Max W. Chang designed and performed all biochemical analyses on the selected protease inhibitors. Max W. Chang performed all docking studies. John H. Elder, Dale L. Boger, and Bruce E. Torbett were involved in the design and interpretation of the results. Michael J. Giffin, Max W. Chang, and Bruce E. Torbett were primarily involved in writing the manuscript. Bruce E.