Cryo-EM structure of human PAPP-A2 and mechanism of substrate recognition.

Pregnancy-Associated Plasma Protein A isoforms, PAPP-A and PAPP-A2, are metalloproteases that cleave insulin-like growth factor binding proteins (IGFBPs) to modulate insulin-like growth factor signaling. The structures of homodimeric PAPP-A in complex with IGFBP5 anchor peptide, and inhibitor proteins STC2 and proMBP have been recently reported. Here, we present the single-particle cryo-EM structure of the monomeric, N-terminal LG, MP, and the M1 domains (with the exception of LNR1/2) of human PAPP-A2 to 3.13 Å resolution. Our structure together with functional studies provides insight into a previously reported patient mutation that inactivates PAPP-A2 in a distal region of the protein. Using a combinational approach, we suggest that PAPP-A2 recognizes IGFBP5 in a similar manner as PAPP-A and show that PAPP-A2 cleaves IGFBP5 less efficiently due to differences in the M2 domain. Overall, our studies characterize the cleavage mechanism of IGFBP5 by PAPP-A2 and shed light onto key differences with its paralog PAPP-A.

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition.

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

Sugar phosphate activation of the stress sensor eIF2B.

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

Complex of human Melanotransferrin and SC57.32 Fab fragment reveals novel interdomain arrangement with ferric N-lobe and open C-lobe.

Melanotransferrin (MTf) is an iron-binding member of the transferrin superfamily that can be membrane-anchored or secreted in serum. On cells, it can mediate transferrin-independent iron uptake and promote proliferation. In serum, it is a transcytotic iron transporter across the blood-brain barrier. MTf has been exploited as a drug delivery carrier to the brain and as an antibody-drug conjugate (ADC) target due to its oncogenic role in melanoma and its elevated expression in triple-negative breast cancer (TNBC). For treatment of TNBC, an MTf-targeting ADC completed a phase I clinical trial (NCT03316794). The structure of its murine, unconjugated Fab fragment (SC57.32) is revealed here in complex with MTf. The MTf N-lobe is in an active and iron-bound, closed conformation while the C-lobe is in an open conformation incompatible with iron binding. This combination of active and inactive domains displays a novel inter-domain arrangement in which the C2 subdomain angles away from the N-lobe. The C2 subdomain also contains the SC57.32 glyco-epitope, which comprises ten protein residues and two N-acetylglucosamines. Our report reveals novel features of MTf and provides a point of reference for MTf-targeting, structure-guided drug design.

Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Accelerating pharmaceutical structure-guided drug design: a successful model.

The impact and value of structure-based drug design to pharmaceutical discovery across the industry are now undeniable, with many break-through therapies on the market that are structure based in nature. Enabling the structural research is the Industrial Macromolecular Crystallography Association-Collaborative Access Team (IMCA-CAT), formed over 25 years ago as a world-class research facility at the synchrotron at Argonne National Laboratory. What makes IMCA-CAT unique is the strategy of the founding consortium to comprehensively provide for the evolving needs of industry in one facility. This includes year-round high-quality data, capabilities that match target portfolios, throughput and capacity that are never limiting, and unfailing security. Here, we illuminate the unique capabilities offered by IMCA-CAT and instruct how all industrial organizations can access this facility.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Characterization of novel small-molecule NRF2 activators: Structural and biochemical validation of stereospecific KEAP1 binding.

BACKGROUND: Semi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. METHODS: We measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. RESULTS: We found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. CONCLUSIONS: Although KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. GENERAL SIGNIFICANCE: This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.

P1-substituted symmetry-based human immunodeficiency virus protease inhibitors with potent antiviral activity against drug-resistant viruses.

Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety.

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.

Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket.

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide(1) is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3-41microM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

Discovery of 3H-benzo[4,5]thieno[3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM) kinases.

Pim-1, Pim-2, and Pim-3 are a family of serine/threonine kinases which have been found to be overexpressed in a variety of hematopoietic malignancies and solid tumors. Benzothienopyrimidinones were discovered as a novel class of Pim inhibitors that potently inhibit all three Pim kinases with subnanomolar to low single-digit nanomolar K(i) values and exhibit excellent selectivity against a panel of diverse kinases. Protein crystal structures of the bound Pim-1 complexes of benzothienopyrimidinones 3b (PDB code 3JYA), 6e (PDB code 3JYO), and 12b (PDB code 3JXW) were determined and used to guide SAR studies. Multiple compounds exhibited potent antiproliferative activity in K562 and MV4-11 cells with submicromolar EC(50) values. For example, compound 14j inhibited the growth of K562 cells with an EC(50) value of 1.7 muM and showed K(i) values of 2, 3, and 0.5 nM against Pim-1, Pim-2, and Pim-3, respectively. These novel Pim kinase inhibitors efficiently interrupted the phosphorylation of Bad in both K562 and LnCaP-Bad cell lines, indicating that their potent biological activities are mechanism-based. The pharmacokinetics of 14j was studied in CD-1 mice and shown to exhibit bioavailability of 76% after oral dosing. ADME profiling of 14j suggested a long half-life in both human and mouse liver microsomes, good permeability, modest protein binding, and no CYP inhibition below 20 muM concentration.

2-Pyridyl P1'-substituted symmetry-based human immunodeficiency virus protease inhibitors (A-792611 and A-790742) with potential for convenient dosing and reduced side effects.

A series of symmetry-based HIV protease inhibitors was designed and synthesized. Modification of the core regiochemistry and stereochemistry significantly affected the potency, metabolic stability, and oral bioavailability of the inhibitors, as did the variation of a pendent arylmethyl P3 group. Optimization led to the selection of two compounds, 10c (A-790742) and 9d (A-792611), for advancement to preclinical studies. Both compounds displayed low nanomolar potency against wild type HIV in the presence of human serum, low rates of metabolism in human liver microsomes, and high oral bioavailability in animal models. The compounds were examined in a preclinical model for the hyperbilirubinemia observed with some HIV PIs, and both exhibited less bilirubin elevation than comparator compounds. X-ray crystallographic analyses of the new cores were used to examine differences in their binding modes. The antiviral activity of the compounds against protease inhibitor resistant strains of HIV was also determined.