Chemical manipulation of an activation/inhibition switch in the nuclear receptor PXR.

Nuclear receptors are ligand-activated transcription factors that can often be useful drug targets. Unfortunately, ligand promiscuity leads to two-thirds of receptors remaining clinically untargeted. PXR is a nuclear receptor that can be activated by diverse compounds to elevate metabolism, negatively impacting drug efficacy and safety. This presents a barrier to drug development because compounds designed to target other proteins must avoid PXR activation while retaining potency for the desired target. This problem could be avoided by using PXR antagonists, but these compounds are rare, and their molecular mechanisms remain unknown. Here, we report structurally related PXR-selective agonists and antagonists and their corresponding co-crystal structures to describe mechanisms of antagonism and selectivity. Structural and computational approaches show that antagonists induce PXR conformational changes incompatible with transcriptional coactivator recruitment. These results guide the design of compounds with predictable agonist/antagonist activities and bolster efforts to generate antagonists to prevent PXR activation interfering with other drugs.

The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activity.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.

Pan-HSP90 ligand binding reveals isoform-specific differences in plasticity and water networks.

Isoforms of heat shock protein 90 (HSP90) fold oncoproteins that facilitate all 10 hallmarks of cancer. However, its promise as a therapeutic target remains unfulfilled as there is still no FDA-approved drug targeting HSP90 in disease. Among the reasons hindering progress are side effects caused by pan-HSP90 inhibition. Selective targeting of the four isoforms is challenging due to high sequence and structural similarity. Surprisingly, while decades of drug discovery efforts have produced almost 400 human HSP90 structures, no single ligand has been structurally characterized across all four human isoforms to date, which could reveal structural differences to achieve selectivity. To better understand the HSP90 landscape relevant for ligand binding and design we take a three-pronged approach. First, we solved the first complete set of structures of a single ligand bound to all four human isoforms. This enabled a systematic comparison of how side-chains and water networks respond to ligand binding across isoforms. Second, we expanded our analysis to publicly available, incomplete isoform-ligand series with distinct ligand chemistry. This highlighted general trends of protein and water mobility that differ among isoforms and impact ligand binding. Third, we further probed the Hsp90α conformational landscape for accommodating a congeneric series containing the purine scaffold common to HSP90 inhibitors. This revealed how minor ligand modifications flip ligand poses and perturb water and protein conformations. Taken together, this work illustrates how a systematic approach can shed new light on an "old" target and reveal hidden isoform-specific accommodations of congeneric ligands that may be exploited in ligand discovery and design.

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

New Design Rules for Developing Potent Cell-Active Inhibitors of the Nucleosome Remodeling Factor (NURF) via BPTF Bromodomain Inhibition.

The nucleosome remodeling factor (NURF) alters chromatin accessibility through interactions with its largest subunit,the bromodomain PHD finger transcription factor BPTF. BPTF is overexpressed in several cancers and is an emerging anticancer target. Targeting the BPTF bromodomain presents a potential strategy for its inhibition and the evaluation of its functional significance; however, inhibitor development for BPTF has lagged behind those of other bromodomains. Here we describe the development of pyridazinone-based BPTF inhibitors. The lead compound, BZ1, possesses a high potency (Kd = 6.3 nM) and >350-fold selectivity over BET bromodomains. We identify an acidic triad in the binding pocket to guide future designs. We show that our inhibitors sensitize 4T1 breast cancer cells to doxorubicin but not BPTF knockdown cells, suggesting a specificity to BPTF. Given the high potency and good physicochemical properties of these inhibitors, we anticipate that they will be useful starting points for chemical tool development to explore the biological roles of BPTF.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.

Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays.

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments.

Structural basis for disassembly of katanin heterododecamers.

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αß-tubulin pool for microtubule dynamics.

Microtubule dynamics and polarity stem from the polymerization of αß-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and ß-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αß-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αß-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αß-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αß-tubulin assembly and maintenance to support microtubule dynamics.

Structural basis for the assembly of the mitotic motor Kinesin-5 into bipolar tetramers.

Chromosome segregation during mitosis depends upon Kinesin-5 motors, which display a conserved, bipolar homotetrameric organization consisting of two motor dimers at opposite ends of a central rod. Kinesin-5 motors crosslink adjacent microtubules to drive or constrain their sliding apart, but the structural basis of their organization is unknown. In this study, we report the atomic structure of the bipolar assembly (BASS) domain that directs four Kinesin-5 subunits to form a bipolar minifilament. BASS is a novel 26-nm four-helix bundle, consisting of two anti-parallel coiled-coils at its center, stabilized by alternating hydrophobic and ionic four-helical interfaces, which based on mutagenesis experiments, are critical for tetramerization. Strikingly, N-terminal BASS helices bend as they emerge from the central bundle, swapping partner helices, to form dimeric parallel coiled-coils at both ends, which are offset by 90°. We propose that BASS is a mechanically stable, plectonemically-coiled junction, transmitting forces between Kinesin-5 motor dimers during microtubule sliding. DOI: http://dx.doi.org/10.7554/eLife.02217.001.

Short-chain fatty acids from periodontal pathogens suppress histone deacetylases, EZH2, and SUV39H1 to promote Kaposi's sarcoma-associated herpesvirus replication.

UNLABELLED: Periodontal pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum produce five different short-chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and downregulate expression of silent information regulator-1 (SIRT1). SCFAs also downregulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone trimethylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS). IMPORTANCE: About 20% of KS patients develop KS lesions first in the oral cavity, while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely, short-chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.

Discovery of antivirulence agents against methicillin-resistant Staphylococcus aureus.

Antivirulence agents inhibit the production of disease-causing virulence factors but are neither bacteriostatic nor bactericidal. Antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300, the most widespread community-associated MRSA strain in the United States, were discovered by virtual screening against the response regulator AgrA, which acts as a transcription factor for the expression of several of the most prominent S. aureus toxins and virulence factors involved in pathogenesis. Virtual screening was followed by similarity searches in the databases of commercial vendors. The small-molecule compounds discovered inhibit the production of the toxins alpha-hemolysin and phenol-soluble modulin α in a dose-dependent manner without inhibiting bacterial growth. These antivirulence agents are small-molecule biaryl compounds in which the aromatic rings either are fused or are separated by a short linker. One of these compounds is the FDA-approved nonsteroidal anti-inflammatory drug diflunisal. This represents a new use for an old drug. Antivirulence agents might be useful in prophylaxis and as adjuvants in antibiotic therapy for MRSA infections.

Kaposi's sarcoma-associated herpesvirus induces rapid release of angiopoietin-2 from endothelial cells.

Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca(2+)) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca(2+) influx and Ang-2 release. Inhibition of Ca(2+) mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.

Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity.

Fusobacterium nucleatum is a Gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA colocalized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and Escherichia coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an 'enabler' for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens.

Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain.

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 angstroms crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel alpha-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the "leucine chain." The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.