Functional implication of the homotrimeric multidomain vacuolar sorting receptor 1 (VSR1) from Arabidopsis thaliana.

The vacuolar sorting receptors (VSRs) are specific to plants and are responsible for sorting and transporting particular proteins from the trans-Golgi network to the vacuole. This process is critically important for various cellular functions, including storing nutrients during seed development. Despite many years of intense studies on VSRs, a complete relation between function and structure has not yet been revealed. Here, we present the crystal structure of the entire luminal region of glycosylated VSR1 from Arabidopsis thaliana (AtVSR1) for the first time. The structure provides insights into the tertiary and quaternary structures of VSR1, which are composed of an N-terminal protease-associated (PA) domain, a unique central region, and one epidermal growth factor (EGF)-like domain followed by two disordered EGF-like domains. The structure of VSR1 exhibits unique characteristics, the significance of which is yet to be fully understood.

Evaluation of novel pyrazol-4-yl pyridine derivatives possessing arylsulfonamide tethers as c-Jun N-terminal kinase (JNK) inhibitors in leukemia cells.

A series of 36 pyrazol-4-yl pyridine derivatives (8a-i, 9a-i, 10a-i, and 11a-i) was designed, synthesized, and evaluated for its antiproliferative activity over NCI-60 cancer cell line panel and inhibitory effect against JNK isoforms (JNK1, JNK2, and JNK3). All the synthesized compounds were tested against the NCI-60 cancer cell line panel. Compounds 11b, 11c, 11g, and 11i were selected to determine their GI50s and exerted a superior potency over the reference standard SP600125 against the tested cell lines. 11c showed a GI50 of 1.28 µM against K562 leukemic cells. Vero cells were used to assess 11c cytotoxicity compared to the tested cancer cells. The target compounds were tested against hJNK isoforms in which compound 11e exhibited the highest potency against JNK isoforms with IC50 values of 1.81, 12.7, and 10.5 nM against JNK1, JNK2, and JNK3, respectively. Kinase profiling of 11e showed higher JNK selectivity in 50 kinase panels. Compounds 11c and 11e showed cell population arrest at the G2/M phase, induced early apoptosis, and slightly inhibited beclin-1 production at higher concentrations in K562 leukemia cells relative to SP600125. NanoBRET assay of 11e showed intracellular JNK1 inhibition with an IC50 of 2.81 µM. Also, it inhibited CYP2D6 and 3A4 with different extent and its hERG activity showed little cardiac toxicity with an IC50 of 4.82 µM. hJNK3 was used as a template to generate the hJNK1 crystal structure to explore the binding mode of 11e (PDB ID: 8ENJ) with a resolution of 2.8 °A and showed a typical type I kinase inhibition against hJNK1. Binding energy scores showed that selectivity of 11e towards JNK1 could be attributed to additional hydrophobic interactions relative to JNK3.

Tumor-derived OBP2A promotes prostate cancer castration resistance.

Androgen deprivation therapy (ADT) is a systemic therapy for advanced prostate cancer (PCa); although most patients initially respond to ADT, almost all cancers eventually develop castration-resistant PCa (CRPC). Currently, most research focuses on castration-resistant tumors, and the role of tumors in remission is almost completely ignored. Here, we report that odorant-binding protein (OBP2A) released from tumors in remission during ADT catches survival factors, such as CXCL15/IL8, to promote PCa cell androgen-independent growth and enhance the infiltration of myeloid-derived suppressor cells (MDSCs) into tumor microenvironment, leading to the emergence of castration resistance. OBP2A knockdown significantly inhibits CRPC and metastatic CRPC development and improves therapeutic efficacy of CTLA-4/PD-1 antibodies. Treatment with OBP2A-binding ligand α-pinene interrupts the function of OBP2A and suppresses CRPC development. Furthermore, α-pinene-conjugated doxorubicin/docetaxel can be specifically delivered to tumors, resulting in improved anticancer efficacy. Thus, our studies establish a novel concept for the emergence of PCa castration resistance and provide new therapeutic strategies for advanced PCa.

Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.

Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of ß-diketone-, ß-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2's reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.

Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B cell reprograming may be scientifically and therapeutically useful, but current approaches limit B cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identify more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and PG16. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B cell repertoires recognizing a key HIV-1 neutralizing epitope.

N-Aromatic-Substituted Indazole Derivatives as Brain-Penetrant and Orally Bioavailable JNK3 Inhibitors.

An indazole/aza-indazole scaffold was developed as a novel chemotype for JNK3 inhibition. Extensive structure activity relationship (SAR) studies utilizing various in vitro and in vivo assays led to potent and highly selective JNK3 inhibitors with good oral bioavailability and high brain penetration. One lead compound, 29, was a potent and selective JNK3 inhibitor (IC50 = 0.005 µM) that had significant inhibition (>80% at 1 µM) to only JNK3 and JNK2 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in the human liver microsome (t 1/2 = 92 min), and was orally bioavailable and brain penetrant (brain/plasma ratio: 56%). The cocrystal structure of 29 in human JNK3 at a 2.1 Å resolution showed that indazole or aza-indazole-based JNK3 inhibitors demonstrated a type I kinase inhibition/binding.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Discovery and Optimization of Selective Inhibitors of Meprin α (Part II).

Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable of selectively inhibiting meprin a, or its close relative meprin b, over numerous other metzincins which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting points for meprin a-specific inhibition through an HTS effort (see part I, preceding paper). Here, in part II, we report further efforts to optimize potency and selectivity. We hope that a hydroxamic acid meprin α inhibitor probe will help define the therapeutic potential for small molecule meprin a inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition.

A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020.

CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome.

Thiophene-Pyrazolourea Derivatives as Potent, Orally Bioavailable, and Isoform-Selective JNK3 Inhibitors.

Potent JNK3 isoform selective inhibitors were developed from a thiophenyl-pyrazolourea scaffold. Through structure activity relationship (SAR) studies utilizing enzymatic and cell-based assays, and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies, potent and highly selective JNK3 inhibitors with oral bioavailability and brain penetrant capability were developed. Inhibitor 17 was a potent and isoform selective JNK3 inhibitor (IC50 = 35 nM), had significant inhibition to only JNK3 in a panel profiling of 374 wild-type kinases, had high potency in functional cell-based assays, had high stability in human liver microsome (t 1/2 = 66 min) and a clean CYP-450 inhibition profile, and was orally bioavailable and brain penetrant. Moreover, cocrystal structures of compounds 17 and 27 in human JNK3 were solved at 1.84 Å, which showed that these JNK3 isoform selective inhibitors bound to the ATP pocket, had interactions in both hydrophobic pocket-I and hydrophobic pocket-II.

Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop.

In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202-216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation.

Structural characterization of a peptoid-inspired conformationally constrained oligomer (PICCO) bound to streptavidin.

A high affinity Streptavidin ligand was mined from a DNA-encoded library of non-peptidic oligimers and characterized structurally.

A Simple and Efficient System for Producing Recombinant Human CXCL8 in Escherichia coli.

Multifunctional pro-inflammatory cytokine CXCL8 is a small peptide of 8-10 kDa in size and it functions as a monomer or dimer. CXCL8 harbors 2 disulfide bonds for its stability. Although production of the CXCL8 protein in a large quantity in both mammalian and bacterial systems has been reported, the processes are complicated and lengthy. Here, we develop a new bacterial expression system for recombinant CXCL8 and simplify the purification system to yield a high amount of protein quickly. The purified CXCL8 protein from our new system develops a crystal structure that is identical to that produced through the mammalian expression system. Thus, we have established a simple and efficient recombinant CXCL8-producing system, which can be easily operated and is suitable to those requiring a large quantity of CXCL8.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Site-Specific Lysine Arylation as an Alternative Bioconjugation Strategy for Chemically Programmed Antibodies and Antibody-Drug Conjugates.

By exploiting a uniquely reactive lysine residue (Lys99) for site-specific attachment of small molecules, the humanized catalytic antibody h38C2 has been used as bioconjugation module in the assembly of chemically programmed antibodies and antibody-drug conjugates. Treatment of h38C2 with ß-lactam-functionalized small molecules has been previously shown to result in covalent conjugation by selective formation of a stable amide bond with the ε-amino group of the Lys99 residue. Here we report that heteroaryl methylsulfonyl (MS-PODA)-functionalized small molecules represent an alternative bioconjugation strategy through highly efficient, site-specific, and stable arylation of the Lys99 residue. A set of chemically programmed antibodies and antibody-drug conjugates assembled by Lys99 arylation provided proof-of-concept for the therapeutic utility of this alternative bioconjugation strategy. While being equally effective as ß-lactam-functionalized ligands for bioconjugation with catalytic antibody h38C2, the MS-PODA moiety offers distinct synthetic advantages, making it highly attractive.

Site-Selective Antibody Functionalization via Orthogonally Reactive Arginine and Lysine Residues.

Homogeneous antibody-drug conjugates (ADCs) that use a highly reactive buried lysine (Lys) residue embedded in a dual variable domain (DVD)-IgG1 format can be assembled with high precision and efficiency under mild conditions. Here we show that replacing the Lys with an arginine (Arg) residue affords an orthogonal ADC assembly that is site-selective and stable. X-ray crystallography confirmed the location of the reactive Arg residue at the bottom of a deep pocket. As the Lys-to-Arg mutation is confined to a single residue in the heavy chain of the DVD-IgG1, heterodimeric assemblies that combine a buried Lys in one arm, a buried Arg in the other arm, and identical light chains, are readily assembled. Furthermore, the orthogonal conjugation chemistry enables the loading of heterodimeric DVD-IgG1s with two different cargos in a one-pot reaction and thus affords a convenient platform for dual-warhead ADCs and other multifaceted antibody conjugates.

HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists.

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it's unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion.

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 Å resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.