A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Identification and characterization of a selective peroxisome proliferator-activated receptor beta/delta (NR1C2) antagonist.

The identification of small molecule ligands for the peroxisome proliferator-activated receptors (PPARs) has been instrumental in elucidating their biological roles. In particular, agonists have been the focus of much of the research in the field with relatively few antagonists being described and all of those being selective for PPARalpha or PPARgamma. The comparison of these agonist and antagonist ligands in cellular and animal systems has often led to surprising results and new insights into the biology of the PPARs. The PPARbeta/delta receptor is emerging as an important regulator of energy metabolism, inflammation, and cell growth and differentiation; however, only agonist ligands have been described for this receptor thus far. Here we describe the first report of a PPARbeta/delta small molecule antagonist ligand. This antagonist ligand will be a useful tool for elucidating the biological roles of PPARbeta/delta.

The nuclear receptor-coactivator interaction surface as a target for peptide antagonists of the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARalpha, PPARdelta, and PPARgamma) constitute a family of nuclear receptors that regulates metabolic processes involved in lipid and glucose homeostasis. Although generally considered to function as ligand-regulated receptors, all three PPARs exhibit a high level of constitutive activity that may result from their stimulation by intracellularly produced endogenous ligands. Consequently, complete inhibition of PPAR signaling requires the development of inverse agonists. However, the currently available small molecule antagonists for the PPARs function only as partial agonists, or their efficacy is not sufficient to inhibit the constitutive activity of these receptors. Due to the lack of efficacious antagonists that interact with the ligand-binding domain of the PPARs, we decided to target an interaction that is central to nuclear receptor-mediated gene transcription: the nuclear receptor-coactivator interaction. We utilized phage display technology to identify short LXXLL-containing peptides that bind to the PPARs. Analysis of these peptides revealed a consensus binding motif consisting of HPLLXXLL. Cross-screening of these peptides for binding to other nuclear receptors enabled the identification of a high-affinity PPAR-selective peptide that has the ability to repress PPARgamma1-dependent transcription of transfected reporter genes. Most importantly, when introduced into HepG2 cells, the peptide inhibited the expression of endogenous PPARgamma1 target genes, adipose differentiation-related protein and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase 2. This work lends support for the rational development of peptidomimetics that block receptor-mediated transcription by targeting the nuclear receptor-coactivator interaction surface.

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening.

A high throughput scintillation proximity assay with biotinylated human immunodeficiency virus (HIV) Rev protein and tritiated Rev response element RNA was used to screen over 500,000 small molecules. Several chemical classes of inhibitors and two chemical classes of enhancers of binding were identified, with the molecular weight range being 400-600. The most common structural motif of inhibitor was an acidic moiety at the end of a linear aromatic system. Most of these modulators had EC(50) values in the 1-10 microM potency range, with several below 1 microM. Several classes displayed structure-activity relationships suggesting specific molecular interactions between small molecule and macromolecule. Several molecules were confirmed as inhibitors in a gel shift assay and by surface plasmon resonance analysis. Furthermore, one inhibitor was shown to bind the Rev protein with a binding constant equal to its IC(50) value, consistent with the mechanism of inhibition being binding Rev. Thus, small molecules can modulate this macromolecular protein-RNA interaction in vitro. However, no compound demonstrated HIV antiviral activity in a relevant cell-based assay.

Characterization of a novel PERK kinase inhibitor with antitumor and antiangiogenic activity.

The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stresses that include hypoxia, nutrient deprivation, and change in redox status. These stress stimuli are common in many tumors and thus targeting components of the UPR signaling is an attractive therapeutic approach. We have identified a first-in-class, small molecule inhibitor of the eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3) or PERK, one of the three mediators of UPR signaling. GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity with an IC(50) of 0.9 nmol/L. It is highly selective for PERK with IC(50) values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells with an IC(50) in the range of 10-30 nmol/L as shown by inhibition of stress-induced PERK autophosphorylation, eIF2α substrate phosphorylation, together with corresponding decreases in ATF4 and CAAT/enhancer binding protein homologous protein (CHOP) in multiple cell lines. Oral administration of GSK2656157 to mice shows a dose- and time-dependent pharmacodynamic response in pancreas as measured by PERK autophosphorylation. Twice daily dosing of GSK2656157 results in dose-dependent inhibition of multiple human tumor xenografts growth in mice. Altered amino acid metabolism, decreased blood vessel density, and vascular perfusion are potential mechanisms for the observed antitumor effect. However, despite its antitumor activity, given the on-target pharmacologic effects of PERK inhibition on pancreatic function, development of any PERK inhibitor in human subjects would need to be cautiously pursued in cancer patients.

Identification and characterization of 4-chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), a selective and irreversible peroxisome proliferator-activated receptor delta (PPARdelta) antagonist.

4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3 (GSK3787) was identified as a potent and selective ligand for PPARdelta with good pharmacokinetic properties. A detailed binding study using mass spectral analysis confirmed covalent binding to Cys249 within the PPARdelta binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARdelta and inhibited basal CPT1a gene transcription. Compound 3 is a PPARdelta antagonist with utility as a tool to elucidate PPARdelta cell biology and pharmacology.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Modulation of androgen receptor activation function 2 by testosterone and dihydrotestosterone.

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.

A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor.

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.

Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin.

Two different sites on vitamin K-dependent gamma-glutamyl carboxylase (VKC) are involved in enzyme-substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-K(m) substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the gamma-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.

Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein.

Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

Subtype specific effects of peroxisome proliferator-activated receptor ligands on corepressor affinity.

Natural ligands for nuclear receptors are believed to activate gene transcription by causing dissociation of corepressors and promoting the association of coactivator proteins. Using multiple biophysical techniques, we find that peptides derived from one of the nuclear receptor interacting motifs of the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT) are able to bind the ligand binding domains (LBD) of all three PPAR (peroxisome proliferator activated receptor) subtypes. Using these peptides as tools, we find that ligands designed as selective agonists for PPAR gamma promote the association of coactivator peptides and dissociation of corepressor peptides as expected on PPAR gamma but surprisingly have varied effects on the binding of corepressor peptides to the other PPAR subtypes. In particular, some members of a class of L-tyrosine-based compounds designed as selective agonists for PPAR gamma reduce the affinity for corepressor peptides on PPAR gamma but increase the affinity for the same peptides on PPAR delta and in one case on PPAR alpha. We provide structural data that suggests that the molecular basis for these observations are variations in the ligand binding pockets of the three PPAR subtypes that are perturbed differentially by individual ligands and result in altered presentations of the overlapping coactivator/corepressor binding surfaces.

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance.

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.

Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.

Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.

Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARalpha.

Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR, which in turn recruit histone deacetylases to the chromatin. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-alpha ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn alpha-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.