Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).

Supplementary Figure 1 from SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

Supplementary Tables 1-3, Figures 1-4 from SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen-binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co-expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co-expression/disassociation methodology on two difficult-to-express antigen ECDs from cluster-of-differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine-tagged and/or Fc-fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.

A retrospective of structural genomics from within the biotech and pharma industry will be presented along with a perspective on the future of structural sciences in the biopharma industry. My fi rst exposure to structural genomics was at the 1998 Avalon, NJ meeting, which initiated my career in structural bioinformatics. From 2000 - 2008 I worked at Structural GenomiX, later SGX Pharmaceuticals, which became a protein production center during the fi rst phase of the NIGMS - funded Protein Structure Initiative, and one of the large scale production centers during PSI-2. During this time, SGX was involved in internal drug discovery research, multiple commercial collaborations, and work with academic and non-profit organizations. In 2008 the company was acquired by Eli Lilly & Company and moved to a new site, all the while continuing its structural genomics efforts. I will provide some perspective on the first “PSI” decade, the following decade focused on using structural data to accelerate pharmaceutical discovery research, and the current decade of artificial intelligence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

Kathryn Westendorf, Stefanie Žentelis, Denisa Foster, Peter Vaillancourt, Matthew Wiggin, Erica Lovett, Jörg Hendle, Anna Pustilnik, J. Michael Sauder, Lucas Kraft, Yuri Hwang, Robert W. Siegel, Jinbiao Chen, Beverly A. Heinz, Richard E. Higgs, Nicole Kalleward, Kevin Jepson, Rodrigo Goya, Maia A. Smith, David W. Collins, Davide Pellacani, Ping Xiang, Valentine de Puyraimond, Marketa Ricicova, Lindsay Devorkin, Caitlin Pritchard, Aoise O’Neill, Courtney Cohen, John Dye, Kathleen I. Huie, Catherine V. Badger, Darwyn Kobasa, Jonathan Audet, Joshua J. Freitas, Saleema Hassanali, Ina Hughes, Luis Munoz, Holly C. Palma, Bharathi Ramamurthy, Robert W. Cross, Thomas W. Geisbert, Viktoriya Borisevich, Iliana Lanz, Lisa Anderson, Payal Sipahimalani, Kizzmekia S. Corbett, Lingshu Wang, Eun Sung Yang, Yi Zhang, Wei Shi, Barney S. Graham, John R. Mascola, Tara L. Fernandez, Carl L. Hansen, Ester Falconer, Bryan E. Jones, and Bryan C. Barnhart

Objective Fibroblast‐like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase‐8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. Methods RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5‐8. Caspase‐8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase‐8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase‐8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase‐8B and G were determined. Results Caspase‐8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase‐8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase‐8) and induced by PDGF. The crystal structures of caspase‐8 variant G and B were identical except for a unique unstructured 59 amino acid N‐terminal domain in variant G. Selective knockdown of caspase‐8G was solely responsible for the effects of caspase‐8 on calpain activity and cell invasion in FLS. Conclusion Blocking caspase‐8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase‐8 inhibition.

G-protein coupled receptors (GPCRs) are the ligand detection machinery of a majority of extracellular signaling systems in metazoans. Novel chemical and biological tools to probe the structure-function relationships of GPCRs have impacted both basic and applied GPCR research. To better understand the structure-function of class B GPCRs, we generated receptor-ligand fusion chimeric proteins that can be activated by exogenous enzyme application. As a prototype, fusion proteins of the glucagon-like peptide-1 receptor (GLP-1R) with GLP-1(7–36) and exendin-4(1–39) peptides incorporating enterokinase-cleavable N-termini were generated. These receptors are predicted to generate fusion protein neo-epitopes upon proteolysis with enterokinase that are identical to the N-termini of GLP-1 agonists. This system was validated by measuring enterokinase-dependent GLP-1R mediated cAMP accumulation, and a structure-activity relationship for both linker length and peptide sequence was observed. Moreover, our results show this approach can be used in physiologically relevant cell systems, as GLP-1R-ligand chimeras were shown to induce glucose-dependent insulin secretion in insulinoma cells upon exposure to enterokinase. This approach suggests new strategies for understanding the structure-function of peptide-binding GPCRs.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.

The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells.Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3.Citation Format: Jaafar N. Haidar, Stephen Antonysamy, Sneha Mathew, Lan Wu, Yi Zhang, Margaret C. Kearins, Leyi Shen, J. Michael Sauder, David Schaer, Kyla E. Driscoll, Michael Kalos. The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2753.

Increasing the success rate and throughput of drug discovery will require efficiency improvements throughout the process that is currently used in the pharmaceutical community, including the crucia...

Single wavelength anomalous dispersion (SAD) utilizing anomalous signal from native sulfurs, or other atoms with Z≤20, generally requires highly redundant data collected with relatively long wavelength X-rays. Here we present experiences with two proteins, where anomalous signal from serendipitously acquired surface bound calcium atoms with data redundancy as low as 10 was utilized to drive de novo structure determination. In both the cases, the calcium atoms were acquired from the crystallization solution, and data collection strategy was not optimized for exploiting the anomalous signal from these scatterers. The X-ray data were collected at 0.98Å in one case and at 1.74Å in the other (wavelength optimized for sulfur but anomalous signal from Ca was exploited for structure solution). Similarly, using a test case, we show that the data collected at ~1.0Å wavelength, where the f" value for sulfur is 0.28e, is sufficient for structure determination using intrinsic sulfur atoms from a strongly diffracting crystal. Interestingly, it was also observed that SHELXD was capable of generating a substructure solution from high-exposure data with a completeness of 70% for low-resolution reflections extending to 3.5Å, with relatively low redundancy (i.e., 6.0 fold). Considering the fact that many crystallization conditions contain anomalous scatterers such as Cl, Ca, Mn etc., it appears that the data from well diffracting crystals should be processed, with anomalous pairs kept separate, so as to detect any fortuitous signal that might be present, which could prove useful in either solving the structure de novo or in accurately assigning surface bound atoms.

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.

A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (2) with kcat/Km values of 4.7 × 10(4) and 5.7 × 10(4) M(-1) s(-1) and l-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 × 10(4) and 2.2 × 10(4) M(-1) s(-1), respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members.

The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, and are separated by diffuse active-site sequence boundaries. We infer a high tolerance of the KDO8PP catalytic platform to amino acid replacements that in turn influence substrate specificity changes and thereby facilitate the divergence in biological function.