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Supplementary Material PDF file - 147K, Supplementary Methods: Immunohistochemistry and magnetic resonance imaging. Supplementary Table 1: Kinase selectivity profiling of GSK2656157. Supplementary Table 2: Gene list for Human UPR array

Supplementary Figures 1-3 PDF file - 245K, Supplementary Figure 1: Dose-dependent pharmacodynamic effect of GSK2656157 on PERK in mouse pancreas. Supplementary Figure 2: Specificity of phospho-PERK antibody in mouse cells. Supplementary Figure 3: Effect of PERK inhibitor on RNA expression in tumor xenografts.

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. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.

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.

A microbiological study was undertaken to assess the risk of infection to a CF patient from a collection of pet reptiles, particularly atypical mycobacteria. This study helped to verify that the reptiles under the care of the CF patient did not harbour bacterial organisms that would normally be pathogenic to CF patients. However, the chronic carriage of Pseudomonas aeruginosa and other pathogens in the CF patient may constitute a greater risk of infection to the animals being handled. Therefore, we recommend stringent infection control precautions by CF patients and their pets, particularly adherence to hand washing and disinfection, when handling the animals, their litter or when working with their immediate environment, to potentially minimize the spread of bacterial and other pathogens from animal to human and vice versa. Detailed risk assessments therefore need to be undertaken by clinicians and veterinarians to detail working models that protect both animals and patients from pathogens originating from the other.

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.

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 L XX LL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR F XX LF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound F XX LF and L XX LL 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 L XX LL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in L XX LL motif binding parallels expansion and functional dominance of the NH 2 -terminal transactivation domain in the steroid receptor subfamily.

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.

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 β 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.

Peptides from the venom ducts of cone snails (genus Conus) contain gamma-carboxyglutamate residues. The gamma-glutamyl carboxylase responsible for this post-translational modification is localized in the microsomal fraction, strictly dependent on vitamin K, activated by ammonium sulfate, and is associated with endogenous substrate. The K(m) of the enzyme for vitamin K is comparable to that for the bovine carboxylase. However, a propeptide containing substrate related to the blood coagulation protein factor IX, a highly efficient substrate for the bovine enzyme, was poorly carboxylated by the Conus enzyme, suggesting differences in gamma-carboxylase recognition signal sequences and/or structural requirements at the carboxylation site.

Studies on the vitamin K-dependent carboxylase are still in their infancy, but the cloning and purification of the enzyme have permitted a number of advances in our understanding of both the molecular biology, and mechanism of the carboxylase. Advances in our knowledge of the molecular biology of the carboxylase include chromosomal location, characterization of messenger RNA transcript(s) and the study of patients with carboxylase mutations. Our understanding of the mechanism of the carboxylase has been enhanced by the expression of peptides in E. coli which contain multiple carboxylation sites and more closely resemble the native carboxylase substrates. These peptides have been utilized to identify elements within the substrates which are critical for carboxylation and to demonstrate that the vitamin K-dependent carboxylase is one of the first examples of a processive post-translational modification enzyme.

We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.

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 IC50 of 0.9 nmol/L. It is highly selective for PERK with IC50 values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells with an IC50 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. Cancer Res; 73(6); 1993–2002. ©2012 AACR.

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.

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 peroxisome proliferator-activated receptors (PPARα, PPARδ, and PPARγ) 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 PPARγ1-dependent transcription of transfected reporter genes. Most importantly, when introduced into HepG2 cells, the peptide inhibited the expression of endogenous PPARγ1 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.

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.

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.

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.

Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR1,2, which in turn recruit histone deacetylases to the chromatin3,4,5. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome6,7,8. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists9. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-α 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 α-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.

Two different sites on vitamin K-dependent γ-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-Km 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 γ-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.

The binding of the gamma-glutamyl carboxylase to its protein substrates is mediated by a conserved 18 amino acid propeptide sequence found in all vitamin K-dependent proteins. We recently found that the apparent affinities of the naturally occurring propeptides for the carboxylase vary over a 100-fold range and that the propeptide of bone Gla protein has severely impaired affinity for the carboxylase [Stanley, T. B., et al. (1999) J. Biol. Chem. 274, 16940-16944 (1)]. Here we report a consensus propeptide sequence that binds tighter (K(i) = 0.43 nM) to the carboxylase than any known propeptide sequence. Comparing the factor IX propeptide to the propeptides of protein C, bone Gla protein, and prothrombin, the weakest binding propeptides, allowed us to predict which residues might be responsible for these substrates' relatively weak binding to the carboxylase. We then made propeptides with the predicted amino acid changes and determined their binding affinities. The reduced binding affinity of these propeptides relative to that of FIX is due to residues -15 in protein C, -10 and -6 in bone Gla protein, and -9 in prothrombin. A role for the -9 position was not previously recognized but is further shown by our identification of a new, naturally occurring mutation at this position in factor IX which causes a warfarin-sensitive hemophilia B phenotype. In addition, we find that propeptides with mutations found in warfarin-sensitive patients have reduced affinity for the carboxylase, suggesting a physiological relevance of propeptide binding affinity.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (Ki) toward a factor IX propeptide/gamma-carboxyglutamic acid domain substrate. The Ki values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are approximately 100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.

On the basis of their tumorigenic properties in hamsters, human adenoviruses have been divided into subgroup A, highly oncogenic, and subgroup B, weakly oncogenic, adenoviruses (1). Recent studies (2–5) have established a third subgroup, C, which consists of types 1, 2, 5 and 6. These types under appropriate conditions transform rat embryo cells, and like the viruses of subgroups A and B they produce a subgroup specific mRNA and a common T antigen. The nonvirion nature of the T antigen of subgroups A and B has been amply documented by immunologic and biophysical methods employing purified antigens (1, 6–9). This report presents similar results on the relationship between T and structural antigens of adenovirus 2, a member of subgroup C. Soluble viral antigens were produced in KB cell monolayers, which were harvested 48 hr after infection with adenovirus 2 (prototype strain).

One of the most petroliferous structural features of the Gulf of Mexico basin is the Vicksburg fault zone of southern Texas and northeastern Mexico. For almost 300 mi the downthrown side of these down-to-basin faults is characterized by an alignment of oil and gas fields, representing a total ultimate reserve of more than 3 billion bbl of oil and 20 trillion ft3 of gas. Although the Vicksburg fault-zone trend has been explored over a period of more than 30 years, it still is not clearly understood. The principal reasons for the lack of understanding are that the displacements are buried deeply and have been interpreted almost entirely from seismic records and well data. The accumulation of the hydrocarbons associated with the fault zone is largely a result of two notable characteristics of the zone: (1) downbending of the downthrown block provided dip reversal necessary for the formation of anticlinal closures; and (2) faulting contemporaneous with deposition resulted in the formation of structural closure during deposition and, thus, in early entrapment of migrating oil and gas. Throughout the zone the Vicksburg faults show a history of post-Eocene movement. However, data collected at widely separated places indicate the possibility of earlier movements. Maximum activity occurred during Oligocene and early Miocene time, when the terrigenous clastic sediments of the Vicksburg and lower Frio were being deposited. Faulting decreased and ceased during or shortly after deposition of the upper Frio. In places, where the fault zone consists of multiple faults, movement shifted progressively from one fault to another.