To identify ligands for the regulatory PHD1 domain of KDM5A, high-throughput screening and biophysical validation were conducted and led to the identification of benzofuran ligands. This work offers a starting point for further optimization of PHD1 ligands.

During the last two decades, two distinct monoclonal antibodies, RP215 and GHR106 were generated, respectively and extensively characterized, biologically and immunologically. Both antibodies target separately specific pan cancer markers and are being evaluated preclinically for potential therapeutic applications in cancer immunotherapy and/or fertility regulations. RP215 was shown to react specifically with carbohydrate-associated epitope located in the heavy chain variable regions of cancer cell expressed specific immunoglobulins, designated as CA215 which are distinct from those of normal B cell origins. The cancerous immunoglobulins may function to react with specific human serum proteins to facilitate growth/proliferation as well as protection of cancer cells in circulations. RP215-based enzyme immunoassays were designed to monitor serum CA215 levels among cancer patients. On the other hand, GHR106 was generated against N1-29 oligopeptide located in the extracellular domains of human GnRH receptor found either in the anterior pituitary or in most of the cancer cells. In vitro culture of cancer cells revealed that either of these two antibodies can induce apoptosis of cancer cells following 24 - 48 hours incubations. Anti-tumor activities of both antibodies were evaluated by typical nude mouse experiments. Either one was shown to effectively reduce the volumes of implanted tumors, dose-dependently. Humanized forms of either antibody were made available in CAR (chimeric antigen receptor)-T cell constructs. They were shown separately to induce cytotoxic killings of cancer cells in vitro by releasing cytokines upon incubations of tumor cells with either of CAR-T cell constructs. In addition, GHR106 also acts as GnRH antagonist by a specific targeting to pituitary GnRH receptor for reversible suppressions of reproductive hormones such as LH, testosterone or estradiol. Based on the above preclinical assessments, it can be generally concluded that both RP215 and GHR106 are restricted in normal tissue expressions and suitable for targeting cancerous immunoglobulins and GnRH receptor, respectively for cancer immunotherapy. Furthermore, specific targeting of pituitary GnRH receptor may suggest that the long acting GHR106 (5 - 21 days half-life) is an adequate GnRH antagonist for numerous gynecological treatments including ovulation inhibition in IVF/ART, endometriosis, premenstrual syndrome, precocious puberty, uterine fibroids and/or polycystic ovarian syndrome.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

Protein-protein interactions (PPIs) occur in complex networks. These networks are highly dependent on cellular context and can be extensively altered in disease states such as cancer and viral infection. In recent years, there has been significant progress in developing inhibitors that target individual PPIs either orthosterically (at the interface) or allosterically. These molecules can now be used as tools to dissect PPI networks. Here, we review recent examples that highlight the use of small molecules and engineered proteins to probe PPIs within the complex networks that regulate protein homeostasis. Researchers have discovered multiple mechanisms to modulate PPIs involved in host/viral interactions, deubiquitinases, the ATPase p97NCP, and HSP70 chaperones. However, few studies have evaluated the effect of such modulators on the target's network or have compared the biological implications of different modulation strategies. Such studies will have an important impact on next generation therapeutics.

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.

, 142 (2005); 309 Science et al. Miles A. Pufall Unstructured Region Variable Control of Ets-1 DNA Binding by Multiple Phosphates in an This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): May 8, 2014 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/309/5731/142.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2005/06/27/309.5731.142.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/309/5731/142.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/309/5731/142.full.html#ref-list-1 , 15 of which can be accessed free: cites 30 articles This article 56 article(s) on the ISI Web of Science cited by This article has been http://www.sciencemag.org/content/309/5731/142.full.html#related-urls 43 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/molec_biol Molecular Biology subject collections: This article appears in the following

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions.

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [N-15]Cys, [N-15]His, and [C-13]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

All members of the human herpesvirus protease (HEW Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-angstrom-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 angstrom from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions. (C) 2011 Elsevier Ltd. All rights reserved.

We have identified a small molecule (DD2) that inhibits an essential dimeric protease of the human Kaposi’s sarcoma-associated herpesvirus (KSHV Pr). Proteolytic activity of KSHV Pr is disrupted by dissociation of dimeric state. Circular dichroism, chemical shift perturbation mapping and hydrogen exchange experiments indicate that a significant loss of structure at the C-terminal helices occurs concurrently with the dimer to monomer transition [1]. Complementary methods including size exclusion chromatography and 2D NMR spectroscopy show that the monomeric protease is enriched in the presence of DD2, leading to loss of enzymatic activity. 1H – 15N HSQC titration studies mapped the inhibitor-binding site to the dimer interface and mutagenesis studies targeting this region confirmed this binding mode [2]. As currently there are no specific treatments for herpesviral infections and with increased incidents of drug resistance, there is a need for the identification of new human herpesvirus therapeutics. The crystal structure of a monomeric form of KSHV Pr in complex with the small molecule inhibitor bound at the dimer interface was solved at 2.0 Å resolution. The final Rfactor/Rfree is 20.5%/24.3%, respectively. The complex was crystallized in the I222 space group. Two monomers of the truncated KSHV Pr were observed in the asymmetric unit with one DD2 molecule bound to monomer A, and two DD2 molecules bound to monomer B. The crystal structure is consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of an inactive monomeric intermediate. This represents the first crystallographic structure of an inhibited monomeric Human Herpesvirus Protease to date.

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2–H5), resembling the SAM domain, appended with two additional helices (H0–H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

Site‐specific pKa values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH‐dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pKa calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue‐specific pKa values since pH‐dependent chemical shift changes can arise from many sources, including through‐bond inductive effects, through‐space electric field effects, and conformational changes. We have re‐measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH‐dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pKa values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by 1H NMR (Bartik et al., Biophys J 1994;66:1180–1184). This analysis gives insights into the true accuracy associated with experimentally measured pKa values. We find that apparent pKa values frequently differ by 0.5–1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pKa values, which reflects the difficulty in fitting and assigning pH‐dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pKa values, for the benchmarking of protein pKa calculation algorithms, and for the understanding of protein electrostatics in general. Proteins 2011. © 2010 Wiley‐Liss, Inc.

We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N HSQC titration studies mapped the inhibitor binding site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small-molecule inhibitors.

We have employed pramlintide (prAM) as a surrogate for hAM in CD and NMR studies of the conformational preferences of the N-terminal portion of the structure in media which do not provide long-lived monomeric solutions of hAM due to its rapid conversion to preamyloid beta aggregate states. Direct comparison of hAM and prAM could be made under helix-formation-favoring conditions. On the basis of CD and NMR studies: (i) the Cys(2)-Cys(7) loop conformation has a short-span of helix (Ala(5)-Cys(7)); (ii) the extent to which this helix propagates further into the sequence is medium-dependent; a helix from Ala(5) through Ser(20) (with end fraying from His(18) onward) is observed in aqueous fluoroalcohol media; (iii) in 12+ vol.% HFIP, the amyloidogenic region of hAM forms a second helical domain (Phe(23)-Ser(29)); (iv) the two helical regions of hAM do not have any specific geometric relationship as they are connected by a flexible loop that takes different conformations and (v) although the extreme C-terminus is essential for bioactivity, it is found to be extensively randomized with conformer interconversions occurring at a much faster rate than that is observed in the remainder of the peptide sequence. Two NMR-derived structures of the 1-22 sequence fragment of hAM have been derived. The work also serves to illustrate improved methods for the NMR characterization of helices. A detailed quantitative analysis of the NOE intensities observed in aqueous HFIP revealed alternative conformations in the C-terminal portion of the common amylin helix, a region that is known to be involved in the biorecognition phenomena leading to amyloidogenesis. Even though the SNN sequence appears to be a flexible loop, the chemical shifts (and changes induced upon helix structuring) suggest some interactions between the loop and the amyloidogenic segment of hAM that occur on partial helix formation.

Background: RP215 was the first generated monoclonal antibody which recognizes specifically carbohydrate-associated epitope(s) localized in the variable region of the cancer cell-expressed immunoglobulin heavy chains, designated in general as CA215. Clinical evaluations were performed to assess if RP215 can be utilized in immunoassays to determine serum levels of CA215 among ovarian or cervical cancer patients for monitoring purposes.Methods: By using immunoassays, serum CA215 levels were determined from cervical or ovarian cancer patients and evaluated based on their respective clinical stages as well as the conditions of clinical treatments.Results: Multi-center clinical trials were performed with frozen serum specimens of patients with ovarian or clinical cancers to determine the efficacy of RP215-based immunoassays. In the case of ovarian cancer, CA215 immunoassay kit has a sensitivity of 46-68% as compared to 46-70% for that of the corresponding CA125 kit. When both markers were combined for simultaneous monitoring of cancer patients, the sensitivity could be as high as 72-87%. Further analysis revealed that serum CA215 levels among cancer patients are well correlated with stages of tumor progression as well as surgical or radio/chemo treatments.Conclusions: The results of these multi-center clinical studies suggest that CA215 is a suitable pan cancer marker for the monitoring of ovarian/cervical cancers and maybe of several others which express CA215 and can be detected in human circulations.

OC-3-VGH ovarian cancer cell line and numerous others from different human tissue origins were studied for their respective expressions of immunoglobulins as well as carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody. With no exceptions, all the cancer cell lines studied so far express human immunoglobulin G (IgG) heavy chains when determined by Western blot or nested RT-PCR with appropriate primers in the constant region. By Western blot assay, it was also shown that greater than 90% of cancer cell lines expressed RP215-specific epitope(s) on the detected heavy chain molecules. Further studies with OC-3-VGH cancer cells revealed the expressions of all immunoglobulin classes, subclasses, heavy as well as light chains. The primary structure of the IgG heavy chains expressed by single cloned cells of this cancer cell line was elucidated. It was shown to be homologous to that of normal human IgG1 heavy chain derived from B cells, except with high content of serine/threonine residues in the variable region. Expressions of other immunoglobulin-related genes were also detected. Widespread expressions of immunoglobulin heavy chains among cancer cells as well as the frequent presence of unique carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody might have important biological implications during carcinogenesis and applications in immunodiagnostics and antibody-based anti-cancer drug developments.

Structure-based drug design traditionally uses static protein models as inspirations for focusing on "active" site targets. Allosteric regulation of biological macromolecules, however, is affected by both conformational and dynamic properties of the protein or protein complex and can potentially lead to more avenues for therapeutic development. We discuss the advantages of searching for molecules that conformationally trap a macromolecule in its inactive state. Although multiple methodologies exist to probe protein dynamics and ligand binding, our current discussion highlights the use of nuclear magnetic resonance spectroscopy in the drug discovery and design process.

Binding of the transcription factor Ets-1 to DNA is allosterically regulated by a serine-rich region (SRR) that modulates the dynamic character of the adjacent structured DNA-binding ETS domain and its flanking autoinhibitory elements. Multi-site phosphorylation of the flexible SRR in response to Ca2+ signaling mediates variable regulation of Ets-1 DNA-binding affinity. In this study, we further investigated the mechanism of this regulation. First, thermal and urea denaturation experiments demonstrated that phosphorylation of the predominantly unstructured SRR imparts enhanced thermodynamic stability on the well-folded ETS domain and its inhibitory module. We next identified a minimal fragment (residues 279–440) that exhibits both enhanced autoinhibition of Ets-1 DNA-binding and allosteric reinforcement by phosphorylation. To test for intramolecular interactions between the SRR and the rest of the fragment that were not detectable by 1H-1H NOE measurements, paramagnetic relaxation enhancements were performed using Cu2+ bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amide and methyl groups revealed a preferential interaction surface for the flexible SRR extending from the inhibitory module to the DNA-binding interface. Phosphorylation enhanced the localization of the SRR to this surface. We therefore hypothesize that the positioning of the SRR at the DNA-binding interface and its role in shifting Ets-1 to an inhibited conformation are linked. In particular, transient interactions dampen the conformational flexibility of the ETS domain and inhibitory module required for high-affinity binding, as well as possibly occlude the DNA interaction site. Surprisingly, the phosphorylation-dependent effects were relatively insensitive to changes in ionic strength, suggesting that electrostatic forces are not the dominant mechanism for mediating these interactions. The results of this study highlight the role of flexibility and transient binding in the variable regulation of Ets-1 activity.