Publications by authors named "Jeanne A Stuckey"

83 Publications

Discovery of EEDi-5273 as an Exceptionally Potent and Orally Efficacious EED Inhibitor Capable of Achieving Complete and Persistent Tumor Regression.

J Med Chem 2021 Oct 6;64(19):14540-14556. Epub 2021 Oct 6.

Ascentage Pharma Group, Suzhou, Jiangsu 215216, China.

Embryonic ectoderm development (EED) is a promising therapeutic target for human cancers and other diseases. We report herein the discovery of exceptionally potent and efficacious EED inhibitors. By conformational restriction of a previously reported EED inhibitor, we obtained a potent lead compound. Further optimization of the lead yielded exceptionally potent EED inhibitors. The best compound EEDi-5273 binds to EED with an IC value of 0.2 nM and inhibits the KARPAS422 cell growth with an IC value of 1.2 nM. It demonstrates an excellent PK and ADME profile, and its oral administration leads to complete and persistent tumor regression in the KARPAS422 xenograft model with no signs of toxicity. Co-crystal structures of two potent EED inhibitors with EED provide a solid structural basis for their high-affinity binding. EEDi-5273 is a promising EED inhibitor for further advanced preclinical development for the treatment of human cancer and other human diseases.
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http://dx.doi.org/10.1021/acs.jmedchem.1c01059DOI Listing
October 2021

Open Reading Frame 1 Protein of the Human Long Interspersed Nuclear Element 1 Retrotransposon Binds Multiple Equivalents of Lead.

J Am Chem Soc 2021 Sep 8;143(37):15271-15278. Epub 2021 Sep 8.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.

The human long interspersed nuclear element 1 (LINE1) has been implicated in numerous diseases and has been suggested to play a significant role in genetic evolution. Open reading frame 1 protein (ORF1p) is one of the two proteins encoded in this self-replicating mobile genetic element, both of which are essential for retrotransposition. The structure of the three-stranded coiled-coil domain of ORF1p was recently solved and showed the presence of tris-cysteine layers in the interior of the coiled-coil that could function as metal binding sites. Here, we demonstrate that ORF1p binds Pb(II). We designed a model peptide, L16CL23C, to mimic two of the ORF1p Cys layers and crystallized the peptide both as the apo-form and in the presence of Pb(II). Structural comparison of the ORF1p with apo-(L16CL23C) shows very similar Cys layers, preorganized for Pb(II) binding. We propose that exposure to heavy metals, such as lead, could influence directly the structural parameters of ORF1p and thus impact the overall LINE1 retrotransposition frequency, directly relating heavy metal exposure to genetic modification.
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http://dx.doi.org/10.1021/jacs.1c06461DOI Listing
September 2021

SD-91 as A Potent and Selective STAT3 Degrader Capable of Achieving Complete and Long-Lasting Tumor Regression.

ACS Med Chem Lett 2021 Jun 10;12(6):996-1004. Epub 2021 May 10.

Rogel Cancer Center, Departments of Internal Medicine and Pharmacology, Medical School, Medicinal Chemistry, College of Pharmacy, Life Sciences Institute, Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States.

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. We report herein our extensive and evaluations of SD-91, the product of the hydrolysis of our previously reported STAT3 degrader SD-36. SD-91 binds to STAT3 protein with a high affinity and displays >300-fold selectivity over other STAT family protein members. SD-91 potently and effectively induces degradation of STAT3 protein and displays a high selectivity over other STAT members and >7000 non-STAT proteins in cells. A single administration of SD-91 selectively depletes STAT3 protein in tumor tissues with a persistent effect. SD-91 achieves complete and long-lasting tumor regression in the MOLM-16 xenograft model in mice even with weekly administration. Hence, SD-91 is a potent, highly selective, and efficacious STAT3 degrader for extensive evaluations for the treatment of human cancers and other diseases for which STAT3 plays a key role.
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http://dx.doi.org/10.1021/acsmedchemlett.1c00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201759PMC
June 2021

Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity.

Nat Commun 2021 05 11;12(1):2621. Epub 2021 May 11.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit neddylation of cullin 3 in cells at low nanomolar concentrations and are 2-3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin neddylation.
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http://dx.doi.org/10.1038/s41467-021-22924-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113459PMC
May 2021

Targeted disruption of pi-pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity.

Sci Rep 2021 01 12;11(1):656. Epub 2021 Jan 12.

Division of Infectious Diseases, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, 48109, USA.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.
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http://dx.doi.org/10.1038/s41598-020-80577-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804308PMC
January 2021

Lipid-based vaccine nanoparticles for induction of humoral immune responses against HIV-1 and SARS-CoV-2.

J Control Release 2021 02 20;330:529-539. Epub 2020 Dec 20.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA; Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.
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http://dx.doi.org/10.1016/j.jconrel.2020.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749995PMC
February 2021

Discovery of Potent Small-Molecule Inhibitors of MLL Methyltransferase.

ACS Med Chem Lett 2020 Jun 14;11(6):1348-1352. Epub 2020 May 14.

Department of Medicinal Chemistry, Department of Internal Medicine, Department of Pharmacology, Department of Pathology, and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States.

The mixed-lineage leukemia (MLL) protein, also known as MLL1, is a lysine methyltransferase specifically responsible for methylation of histone 3 lysine 4. MLL has been pursued as an attractive therapeutic target for the treatment of acute leukemia carrying the MLL fusion gene or MLL leukemia. Herein, we report the design, synthesis, and evaluation of an -adenosylmethionine-based focused chemical library which led to the discovery of potent small-molecule inhibitors directly targeting the MLL SET domain. Determination of cocrystal structures for a number of these MLL inhibitors reveals that they adopt a unique binding mode that locks the MLL SET domain in an open, inactive conformation.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294728PMC
June 2020

Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys) template mediated strategy.

Nat Chem 2020 04 2;12(4):405-411. Epub 2020 Mar 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(II)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(II)AB heterotrimers, while d sites provide pure Pb(II)CD or Pb(II)CD scaffolds. Altering the metal from Pb(II) to Hg(II) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(II)(histidine)(HO) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.
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http://dx.doi.org/10.1038/s41557-020-0423-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169047PMC
April 2020

Discovery and Characterization of 2,5-Substituted Benzoic Acid Dual Inhibitors of the Anti-apoptotic Mcl-1 and Bfl-1 Proteins.

J Med Chem 2020 03 14;63(5):2489-2510. Epub 2020 Feb 14.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona 85724, United States.

Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compound , which binds both Mcl-1 and Bfl-1 with values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024114PMC
March 2020

Structure-Based Discovery of SD-36 as a Potent, Selective, and Efficacious PROTAC Degrader of STAT3 Protein.

J Med Chem 2019 12 10;62(24):11280-11300. Epub 2019 Dec 10.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and an attractive therapeutic target for cancer and other human diseases. Despite 20 years of persistent research efforts, targeting STAT3 has been very challenging. We report herein the structure-based discovery of potent small-molecule STAT3 degraders based upon the proteolysis targeting chimera (PROTAC) concept. We first designed SI-109 as a potent, small-molecule inhibitor of the STAT3 SH2 domain. Employing ligands for cereblon/cullin 4A E3 ligase and SI-109, we obtained a series of potent PROTAC STAT3 degraders, exemplified by SD-36. SD-36 induces rapid STAT3 degradation at low nanomolar concentrations in cells and fails to degrade other STAT proteins. SD-36 achieves nanomolar cell growth inhibitory activity in leukemia and lymphoma cell lines with high levels of phosphorylated STAT3. A single dose of SD-36 results in complete STAT3 protein degradation in xenograft tumor tissue and normal mouse tissues. SD-36 achieves complete and long-lasting tumor regression in the Molm-16 xenograft tumor model at well-tolerated dose-schedules. SD-36 is a potent, selective, and efficacious STAT3 degrader.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01530DOI Listing
December 2019

Structure of the zinc-finger antiviral protein in complex with RNA reveals a mechanism for selective targeting of CG-rich viral sequences.

Proc Natl Acad Sci U S A 2019 11 12;116(48):24303-24309. Epub 2019 Nov 12.

Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CG-enriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA.
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http://dx.doi.org/10.1073/pnas.1913232116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883784PMC
November 2019

A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo.

Cancer Cell 2019 11;36(5):498-511.e17

Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA; Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA; Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.
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http://dx.doi.org/10.1016/j.ccell.2019.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880868PMC
November 2019

How Outer Coordination Sphere Modifications Can Impact Metal Structures in Proteins: A Crystallographic Evaluation.

Chemistry 2019 May 25;25(27):6773-6787. Epub 2019 Apr 25.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA.

A challenging objective of de novo metalloprotein design is to control of the outer coordination spheres of an active site to fine tune metal properties. The well-defined three stranded coiled coils, TRI and CoilSer peptides, are used to address this question. Substitution of Cys for Leu yields a thiophilic site within the core. Metals such as Hg , Pb , and As result in trigonal planar or trigonal pyramidal geometries; however, spectroscopic studies have shown that Cd forms three-, four- or five-coordinate Cd S (OH ) (in which x=0-2) when the outer coordination spheres are perturbed. Unfortunately, there has been little crystallographic examination of these proteins to explain the observations. Here, the high-resolution X-ray structures of apo- and mercurated proteins are compared to explain the modifications that lead to metal coordination number and geometry variation. It reveals that Ala substitution for Leu opens a cavity above the Cys site allowing for water excess, facilitating Cd S (OH ). Replacement of Cys by Pen restricts thiol rotation, causing a shift in the metal-binding plane, which displaces water, forming Cd S . Residue d-Leu, above the Cys site, reorients the side chain towards the Cys layer, diminishing the space for water accommodation yielding Cd S , whereas d-Leu below opens more space, allowing for equal Cd S (OH ) and Cd S (OH ) . These studies provide insights into how to control desired metal geometries in metalloproteins by using coded and non-coded amino acids.
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http://dx.doi.org/10.1002/chem.201806040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510599PMC
May 2019

Structure-Based Design of N-(5-Phenylthiazol-2-yl)acrylamides as Novel and Potent Glutathione S-Transferase Omega 1 Inhibitors.

J Med Chem 2019 03 22;62(6):3068-3087. Epub 2019 Feb 22.

Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy , Sichuan University , No. 17 People's South Road , Chengdu 610041 , P. R. China.

Using reported glutathione S-transferase omega 1 (GSTO1-1) cocrystal structures, we designed and synthesized acrylamide-containing compounds that covalently bind to Cys32 on the catalytic site. Starting from a thiazole derivative 10 (GSTO1-1 IC = 0.6 μM), compound 18 was synthesized and cocrystallized with GSTO1. Modification on the amide moiety of hit compound 10 significantly increased the GSTO1-1 inhibitory potency. We solved the cocrystal structures of new derivatives, 37 and 44, bearing an amide side chain bound to GSTO1. These new structures showed a reorientation of the phenyl thiazole core of inhibitors, 37 and 44, when compared to 18. Guided by the cocrystal structure of GSTO1:44, analogue 49 was designed, resulting in the most potent GSTO1-1 inhibitor (IC = 0.22 ± 0.02 nM) known to date. We believe that our data will form the basis for future studies of developing GSTO1-1 as a new drug target for cancer therapy.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01960DOI Listing
March 2019

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein.

Inorg Chem 2018 Oct 18;57(19):12291-12302. Epub 2018 Sep 18.

LCM, CNRS, Ecole Polytechnique , Université Paris-Saclay , 91128 Palaiseau Cedex , France.

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, αDChC2. (1) While this design demonstrated that a β-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide αD was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDαD (GRαD) with Δ G = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GRαD (a first for an αD peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRαDChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs' hyperfine coupling constant from 160 to 127 × 10 cm. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41-Cu interaction leads to the αDChC2 construct's red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.
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http://dx.doi.org/10.1021/acs.inorgchem.8b01989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6183058PMC
October 2018

Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H.

J Am Chem Soc 2018 09 10;140(37):11661-11673. Epub 2018 Sep 10.

Department of Chemistry , Case Western Reserve University , Cleveland , Ohio 44106 , United States.

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.
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http://dx.doi.org/10.1021/jacs.8b05366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648666PMC
September 2018

From proteomics to discovery of first-in-class ST2 inhibitors active in vivo.

JCI Insight 2018 07 26;3(14). Epub 2018 Jul 26.

Department of Pediatrics and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.
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http://dx.doi.org/10.1172/jci.insight.99208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124397PMC
July 2018

Structure-Based Discovery of CF53 as a Potent and Orally Bioavailable Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitor.

J Med Chem 2018 07 17;61(14):6110-6120. Epub 2018 Jul 17.

We report the structure-based discovery of CF53 (28) as a highly potent and orally active inhibitor of bromodomain and extra-terminal (BET) proteins. By the incorporation of a NH-pyrazole group into the 9H-pyrimido[4,5- b]indole core, we identified a series of compounds that bind to BRD4 BD1 protein with K values of <1 nM and achieve low nanomolar potencies in the cell growth inhibition of leukemia and breast cancer cells. The most-promising compound, CF53, possesses excellent oral pharmacokinetic properties and achieves significant antitumor activity in both triple-negative breast cancer and acute leukemia xenograft models in mice. Determination of the co-crystal structure of CF53 with the BRD4 BD1 protein provides a structural basis for its high binding affinity to BET proteins. CF53 is very selective over non-BET bromodomain-containing proteins. These data establish CF53 as a potent, selective, and orally active BET inhibitor, which warrants further evaluation for advanced preclinical development.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489120PMC
July 2018

Discovery of Mcl-1 inhibitors from integrated high throughput and virtual screening.

Sci Rep 2018 07 5;8(1):10210. Epub 2018 Jul 5.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA.

Protein-protein interactions (PPIs) represent important and promising therapeutic targets that are associated with the regulation of various molecular pathways, particularly in cancer. Although they were once considered "undruggable," the recent advances in screening strategies, structure-based design, and elucidating the nature of hot spots on PPI interfaces, have led to the discovery and development of successful small-molecule inhibitors. In this report, we are describing an integrated high-throughput and computational screening approach to enable the discovery of small-molecule PPI inhibitors of the anti-apoptotic protein, Mcl-1. Applying this strategy, followed by biochemical, biophysical, and biological characterization, nineteen new chemical scaffolds were discovered and validated as Mcl-1 inhibitors. A novel series of Mcl-1 inhibitors was designed and synthesized based on the identified difuryl-triazine core scaffold and structure-activity studies were undertaken to improve the binding affinity to Mcl-1. Compounds with improved in vitro binding potency demonstrated on-target activity in cell-based studies. The obtained results demonstrate that structure-based analysis complements the experimental high-throughput screening in identifying novel PPI inhibitor scaffolds and guides follow-up medicinal chemistry efforts. Furthermore, our work provides an example that can be applied to the analysis of available screening data against numerous targets in the PubChem BioAssay Database, leading to the identification of promising lead compounds, fuelling drug discovery pipelines.
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http://dx.doi.org/10.1038/s41598-018-27899-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033896PMC
July 2018

Design, Synthesis, and Biological Evaluation of 4-Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase.

J Med Chem 2018 06 14;61(12):5162-5186. Epub 2018 May 14.

Life Sciences Institute and Department of Biological Chemistry , University of Michigan , Ann Arbor , Michigan 48109 , United States.

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC = 9.71 ± 1.4 nM) and 43 (DHODH IC = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01862DOI Listing
June 2018

Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes.

J Biol Chem 2018 02 20;293(6):2125-2136. Epub 2017 Dec 20.

From the Chemical Biology Program,

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of the TNBC cell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis.
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http://dx.doi.org/10.1074/jbc.M117.811463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808772PMC
February 2018

Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction.

J Med Chem 2017 06 12;60(12):4818-4839. Epub 2017 Jun 12.

Department of Medicinal Chemistry, ‡Department of Internal Medicine, §Comprehensive Cancer Center, ∥Department of Pathology, ⊥Howard Hughes Medical Institute, #Department of Biological Chemistry, ∇Department of Pharmaceutical Sciences, ○Department of Pharmacology, and ¶Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.

We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5-mixed lineage leukemia (MLL) protein-protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC value of 0.90 nM (K value <1 nM) and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC value of 12.7 nM. Compound 18 potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations and is >40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5-MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01796DOI Listing
June 2017

Structure-Based Discovery of 4-(6-Methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (CD161) as a Potent and Orally Bioavailable BET Bromodomain Inhibitor.

J Med Chem 2017 05 2;60(9):3887-3901. Epub 2017 May 2.

Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.

We have designed and synthesized 9H-pyrimido[4,5-b]indole-containing compounds to obtain potent and orally bioavailable BET inhibitors. By incorporation of an indole or a quinoline moiety to the 9H-pyrimido[4,5-b]indole core, we identified a series of small molecules showing high binding affinities to BET proteins and low nanomolar potencies in inhibition of cell growth in acute leukemia cell lines. One such compound, 4-(6-methoxy-2-methyl-4-(quinolin-4-yl)-9H-pyrimido[4,5-b]indol-7-yl)-3,5-dimethylisoxazole (31) has excellent microsomal stability and good oral pharmacokinetics in rats and mice. Orally administered, 31 achieves significant antitumor activity in the MV4;11 leukemia and MDA-MB-231 triple-negative breast cancer xenograft models in mice. Determination of the cocrystal structure of 31 with BRD4 BD2 provides a structural basis for its high binding affinity to BET proteins. Testing its binding affinities against other bromodomain-containing proteins shows that 31 is a highly selective inhibitor of BET proteins. Our data show that 31 is a potent, selective, and orally active BET inhibitor.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993113PMC
May 2017

d-Cysteine Ligands Control Metal Geometries within De Novo Designed Three-Stranded Coiled Coils.

Chemistry 2017 Jun 26;23(34):8232-8243. Epub 2017 May 26.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA.

Although metal ion binding to naturally occurring l-amino acid proteins is well documented, understanding the impact of the opposite chirality (d-)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d-configuration cysteine within a designed l-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side-chain re-orientation modifies the sterics of the metal binding pocket. l-Cys side chains within the coiled-coil structure have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by X-ray crystallography that d-Cys side chains are preorganized within a suitable geometry to bind such a ligand. This is confirmed by comparison of the structure of Zn Cl(CSL16 C) to the published structure of Zn (H O)(GRAND-CSL12AL16 C) . Moreover, spectroscopic analysis indicates that the Cd geometry observed by using l-Cys ligands (a mixture of three- and four-coordinate Cd ) is altered to a single four-coordinate species when d-Cys is present. This work opens a new avenue for the control of the metal site environment in man-made proteins, by simply altering the binding ligand with its mirror-imaged d configuration. Thus, the use of non-coded amino acids in the coordination sphere of a metal promises to be a powerful tool for controlling the properties of future metalloproteins.
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http://dx.doi.org/10.1002/chem.201700660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518473PMC
June 2017

Buried Hydrogen Bond Interactions Contribute to the High Potency of Complement Factor D Inhibitors.

ACS Med Chem Lett 2016 Dec 13;7(12):1092-1096. Epub 2016 Sep 13.

Departments of Internal Medicine and Biological Chemistry and Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.

Aberrant activation of the complement system is associated with diseases, including paroxysmal nocturnal hemoglobinuria and age-related macular degeneration. Complement factor D is the rate-limiting enzyme for activating the alternative pathway in the complement system. Recent development led to a class of potent amide containing pyrrolidine derived factor D inhibitors. Here, we used biochemical enzymatic and biolayer interferometry assays to demonstrate that the amide group improves the inhibitor potency by more than 80-fold. Our crystal structures revealed buried hydrogen bond interactions are important. Molecular orbital analysis from quantum chemistry calculations dissects the chemical groups participating in these interactions. Free energy calculation supports the differential contributions of the amide group to the binding affinities of these inhibitors. Cell-based hemolysis assay confirmed these compounds inhibit factor D mediated complement activation via the alternative pathway. Our study highlights the important interactions contributing to the high potency of factor D inhibitors reported recently.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5151139PMC
December 2016

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2).

ACS Chem Biol 2016 12 31;11(12):3374-3382. Epub 2016 Oct 31.

Program in Chemical Biology, ‡Department of Chemistry, §Department of Biophysics, and ∥Life Sciences Institute, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109, United States.

Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.
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http://dx.doi.org/10.1021/acschembio.6b00720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359770PMC
December 2016

D3R grand challenge 2015: Evaluation of protein-ligand pose and affinity predictions.

J Comput Aided Mol Des 2016 09 30;30(9):651-668. Epub 2016 Sep 30.

Drug Design Data Resource, Center for Research in Biological Systems, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA.

The Drug Design Data Resource (D3R) ran Grand Challenge 2015 between September 2015 and February 2016. Two targets served as the framework to test community docking and scoring methods: (1) HSP90, donated by AbbVie and the Community Structure Activity Resource (CSAR), and (2) MAP4K4, donated by Genentech. The challenges for both target datasets were conducted in two stages, with the first stage testing pose predictions and the capacity to rank compounds by affinity with minimal structural data; and the second stage testing methods for ranking compounds with knowledge of at least a subset of the ligand-protein poses. An additional sub-challenge provided small groups of chemically similar HSP90 compounds amenable to alchemical calculations of relative binding free energy. Unlike previous blinded Challenges, we did not provide cognate receptors or receptors prepared with hydrogens and likewise did not require a specified crystal structure to be used for pose or affinity prediction in Stage 1. Given the freedom to select from over 200 crystal structures of HSP90 in the PDB, participants employed workflows that tested not only core docking and scoring technologies, but also methods for addressing water-mediated ligand-protein interactions, binding pocket flexibility, and the optimal selection of protein structures for use in docking calculations. Nearly 40 participating groups submitted over 350 prediction sets for Grand Challenge 2015. This overview describes the datasets and the organization of the challenge components, summarizes the results across all submitted predictions, and considers broad conclusions that may be drawn from this collaborative community endeavor.
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http://dx.doi.org/10.1007/s10822-016-9946-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562487PMC
September 2016

Mechanistic evaluation and transcriptional signature of a glutathione S-transferase omega 1 inhibitor.

Nat Commun 2016 10 5;7:13084. Epub 2016 Oct 5.

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033, USA.

Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we show that silencing of GSTO1 with siRNA significantly impairs cancer cell viability, validating GSTO1 as a potential new target in oncology. We report on the development and characterization of a series of chloroacetamide-containing potent GSTO1 inhibitors. Co-crystal structures of GSTO1 with our inhibitors demonstrate covalent binding to the active site cysteine. These potent GSTO1 inhibitors suppress cancer cell growth, enhance the cytotoxic effects of cisplatin and inhibit tumour growth in colon cancer models as single agent. Bru-seq-based transcription profiling unravelled novel roles for GSTO1 in cholesterol metabolism, oxidative and endoplasmic stress responses, cytoskeleton and cell migration. Our findings demonstrate the therapeutic utility of GSTO1 inhibitors as anticancer agents and identify the novel cellular pathways under GSTO1 regulation in colorectal cancer.
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http://dx.doi.org/10.1038/ncomms13084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059489PMC
October 2016

High-resolution crystal structures of Colocasia esculenta tarin lectin.

Glycobiology 2017 01 24;27(1):50-56. Epub 2016 Aug 24.

Department of Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA

Tarin, the Colocasia esculenta lectin from the superfamily of α-d-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical β-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal β-strand and the β-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYxWX) on face III of the β-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other β-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family.
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http://dx.doi.org/10.1093/glycob/cww083DOI Listing
January 2017

A Crystallographic Examination of Predisposition versus Preorganization in de Novo Designed Metalloproteins.

J Am Chem Soc 2016 09 2;138(36):11979-88. Epub 2016 Sep 2.

Department of Chemistry, ‡Biophysics Program, and §Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.

Preorganization and predisposition are important molecular recognition concepts exploited by nature to obtain site-specific and selective metal binding to proteins. While native structures containing an MS3 core are often unavailable in both apo- and holo-forms, one can use designed three-stranded coiled coils (3SCCs) containing tris-thiolate sites to evaluate these concepts. We show that the preferred metal geometry dictates the degree to which the cysteine rotamers change upon metal complexation. The Cys ligands in the apo-form are preorganized for binding trigonal pyramidal species (Pb(II)S3 and As(III)S3) in an endo conformation oriented toward the 3SCC C-termini, whereas the cysteines are predisposed for trigonal planar Hg(II)S3 and 4-coordinate Zn(II)S3O structures, requiring significant thiol rotation for metal binding. This study allows assessment of the importance of protein fold and side-chain reorientation for achieving metal selectivity in human retrotransposons and metalloregulatory proteins.
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http://dx.doi.org/10.1021/jacs.6b07165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242185PMC
September 2016
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