I03-Macromolecular Crystallography
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Maria Giulia
Nizi
,
Mirko M.
Maksimainen
,
Sudarshan
Murthy
,
Serena
Massari
,
Juho
Alaviuhkola
,
Barbara E.
Lippok
,
Sven T.
Sowa
,
Albert
Galera-Prat
,
Renata
Prunskaite-Hyyryläinen
,
Bernhard
Lüscher
,
Patricia
Korn
,
Lari
Lehtio
,
Oriana
Tabarrini
Diamond Proposal Number(s):
[19951]
Abstract: While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130–160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.
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Jul 2022
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I04-Macromolecular Crystallography
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Jason G.
Kettle
,
Sharan K.
Bagal
,
Sue
Bickerton
,
Michael S.
Bodnarchuk
,
Scott
Boyd
,
Jason
Breed
,
Rodrigo J.
Carbajo
,
Doyle J.
Cassar
,
Atanu
Chakraborty
,
Sabina
Cosulich
,
Iain
Cumming
,
Michael
Davies
,
Nichola L.
Davies
,
Andrew
Eatherton
,
Laura
Evans
,
Lyman
Feron
,
Shaun
Fillery
,
Emma S.
Gleave
,
Frederick W.
Goldberg
,
Lyndsey
Hanson
,
Stephanie
Harlfinger
,
Martin
Howard
,
Rachel
Howells
,
Anne
Jackson
,
Paul
Kemmitt
,
Gillian
Lamont
,
Scott
Lamont
,
Hilary J.
Lewis
,
Libin
Liu
,
Michael J.
Niedbala
,
Christopher
Phillips
,
Radek
Polanski
,
Piotr
Raubo
,
Graeme
Robb
,
David M.
Robinson
,
Sarah
Ross
,
Matthew G.
Sanders
,
Michael
Tonge
,
Rebecca
Whiteley
,
Stephen
Wilkinson
,
Junsheng
Yang
,
Wenman
Zhang
Diamond Proposal Number(s):
[20015]
Abstract: KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure–activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
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May 2022
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan C.
Miller
,
Tristan
Reuillon
,
Lauren
Molyneux
,
Timothy
Blackburn
,
Simon J.
Cook
,
Noel
Edwards
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Ian
Hardcastle
,
Suzannah J.
Harnor
,
Amy
Heptinstall
,
Pamela
Lochhead
,
Mathew P.
Martin
,
Nick C.
Martin
,
Stephanie
Myers
,
David R.
Newell
,
Richard A.
Noble
,
Nicole
Phillips
,
Laurent
Rigoreau
,
Huw
Thomas
,
Julie A.
Tucker
,
Lan-Zhen
Wang
,
Michael J.
Waring
,
Ai-Ching
Wong
,
Stephen R.
Wedge
,
Martin E. M.
Noble
,
Celine
Cano
Diamond Proposal Number(s):
[9948, 13587]
Abstract: The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
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Apr 2022
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I03-Macromolecular Crystallography
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Mark F.
Maurer
,
Katherine E.
Lewis
,
Joseph L.
Kuijper
,
Dan
Ardourel
,
Chelsea J.
Gudgeon
,
Siddarth
Chandrasekaran
,
Sherri L.
Mudri
,
Kayla N.
Kleist
,
Chris
Navas
,
Martin F.
Wolfson
,
Mark W.
Rixon
,
Ryan
Swanson
,
Stacey R.
Dillon
,
Steven D.
Levin
,
Yengo Raymond
Kimbung
,
Masato
Akutsu
,
Derek T.
Logan
,
Björn
Walse
,
Kristine M.
Swiderek
,
Stanford L.
Peng
Diamond Proposal Number(s):
[20028]
Open Access
Abstract: Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4–CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.
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Apr 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[23943]
Abstract: Titanocene dichloride (TDC) is an anticancer agent that delivers Ti(IV) into each of the two Fe(III) binding sites of bilobal human serum transferrin (Tf). This protein has been implicated in the selective transport of Ti(IV) to cells. How Ti(IV) might be released from the Tf Fe(III) binding site has remained a question, and crystal structures have raised issues about lobe occupancy and lobe closure in Ti(IV)-loaded Tf, compared with the Fe(III)-loaded form. Here, inductively coupled plasma optical emission spectroscopy reveals that Tf can stabilize toward hydrolytic precipitation more than 2 equiv of Ti, implying superstoichiometric binding beyond the two Fe(III) binding sites. Further studies support the inability of TDC to induce a complete lobe closure of Tf. Fluorescence data for TDC binding at low equivalents of TDC support an initial protein conformational change and lobe closure upon Ti binding, whereas data at higher equivalents support an open lobe configuration. Spectroscopic titration reveals less intense protein–metal electronic transitions as TDC equivalents are increased. Denaturing urea-PAGE gels and small angle X-ray scattering studies support an open lobe conformation. The concentrations of bicarbonate used in some earlier studies are demonstrated here to cause a pH change over time, which may contribute to variation in the apparent molar absorptivity associated with Ti(IV) binding in the Fe binding site. Finally, Fe(III)-bound holo-Tf still stabilizes TDC toward hydrolytic precipitation, a finding that underscores the importance of the interactions of Tf and TDC outside the Fe(III) binding site and suggests possible new pathways of Ti introduction to cells.
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Apr 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19248]
Open Access
Abstract: The hypoxic response is central to cell function and plays a significant role in the growth and survival of solid tumours. HIF-1 regulates the hypoxic response by activating over 100 genes responsible for adaptation to hypoxia, making it a potential target for anticancer drug discovery. Although there is significant structural and mechanistic understanding of the interaction between HIF-1α and p300 alongside negative regulators of HIF-1α such as CITED2, there remains a need to further understand the sequence determinants of binding. In this work we use a combination of protein expression, chemical synthesis, fluorescence anisotropy and isothermal titration calorimetry for HIF-1α sequence variants and a HIF-1α-CITED hybrid sequence which we term CITIF. We show the HIF-1α sequence is highly tolerant to sequence variation through reduced enthalpic and less unfavourable entropic contributions, These data imply backbone as opposed to side chain interactions and ligand folding control the binding interaction and that sequence variations are tolerated as a result of adopting a more disordered bound interaction or “fuzzy” complex.
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Apr 2022
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B18-Core EXAFS
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Fabio
Del Bello
,
Maura
Pellei
,
Luca
Bagnarelli
,
Carlo
Santini
,
Gianfabio
Giorgioni
,
Alessandro
Piergentili
,
Wilma
Quaglia
,
Chiara
Battocchio
,
Giovanna
Iucci
,
Irene
Schiesaro
,
Carlo
Meneghini
,
Iole
Venditti
,
Nitya
Ramanan
,
Michele
De Franco
,
Paolo
Sgarbossa
,
Cristina
Marzano
,
Valentina
Gandin
Diamond Proposal Number(s):
[25674]
Open Access
Abstract: Bis(pyrazol-1-yl)- and bis(3,5-dimethylpyrazol-1-yl)-acetates were conjugated with the 2-hydroxyethylester and 2-aminoethylamide derivatives of the antineoplastic drug lonidamine to prepare Cu(I) and Cu(II) complexes that might act through synergistic mechanisms of action due to the presence of lonidamine and copper in the same chemical entity. Synchrotron radiation-based complementary techniques [X-ray photorlectron spectroscopy and near-edge X-ray absorption fine structure (NEXAFS)] were used to characterize the electronic and molecular structures of the complexes and the local structure around the copper ion (XAFS) in selected complexes. All complexes showed significant antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human tumor cell lines, and were able to overcome oxaliplatin and multidrug resistance. Noticeably, these Cu complexes appeared much more effective than cisplatin against 3D spheroids of pancreatic PSN-1 cancer cells; among these, PPh3-containing Cu(I) complex 15 appeared to be the most promising derivative. Mechanistic studies revealed that 15 induced cancer cell death by means of an apoptosis-alternative cell death.
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Mar 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Background: The epidermal growth factor receptor (EGFR) is involved in various developmental processes, and alterations of its extracellular segment are associated with several types of cancers, in particular glioblastoma multiforme (GBM). The EGFR extracellular region is therefore a primary target for therapeutic agents, such as monoclonal antibodies and variable domains of heavy chain antibodies (VHH), also called nanobodies. Nanobodies have been previously shown to bind to EGFR, and to inhibit ligand-mediated EGFR activation. Results: Here we present the X-ray crystal structures of the EgB4 nanobody, alone (to 1.48 Å resolution) and bound to the full extracellular EGFR-EGF complex in its active conformation (to 6.0 Å resolution). We show that EgB4 binds to a new epitope located on EGFR domains I and II, and we describe the molecular mechanism by which EgB4 plays a non-inhibitory role in EGFR signaling. Conclusion: This work provides the structural basis for the application of EgB4 as a tool for research, for targeted therapy, or as a biomarker to locate EGFR-associated tumors, all without affecting EGFR activation.
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Mar 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Emily A.
Bates
,
James A.
Davies
,
Jana
Váňová
,
Davor
Nestić
,
Valerie S.
Meniel
,
Sarah
Koushyar
,
Tabitha G.
Cunliffe
,
Rosie M.
Mundy
,
Elise
Moses
,
Hanni K.
Uusi-Kerttula
,
Alexander T.
Baker
,
David K.
Cole
,
Dragomira
Majhen
,
Pierre J.
Rizkallah
,
Toby
Phesse
,
John D.
Chester
,
Alan L.
Parker
Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin selective peptide, A20, to target αvβ6 positive tumour cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation Factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines, demonstrated significantly increased transduction mediated by αvβ6 targeted variants compared to controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumours. Replication competent HAdV-D10.A20 demonstrated αvβ6 integrin selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions and reduced off-target uptake. Incorporation of an αvβ6 integrin selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.
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Mar 2022
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I18-Microfocus Spectroscopy
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Sarah
Gosling
,
Doriana
Calabrese
,
Jayakrupakar
Nallala
,
Charlene
Greenwood
,
Sarah
Pinder
,
Lorraine
King
,
Jeffrey
Marks
,
Donna
Pinto
,
Thomas
Lynch
,
Iain D.
Lyburn
,
E. Shelley
Hwang
,
Cruk
Grand Challenge Precision Consortium
,
Keith
Rogers
,
Nicholas
Stone
Diamond Proposal Number(s):
[21565, 25414, 27300]
Open Access
Abstract: Ductal carcinoma in situ (DCIS) is frequently associated with breast calcification. This study combines
multiple analytical techniques to investigate the heterogeneity of these calcifications at the micrometre
scale. X-ray diffraction, scanning electron microscopy and Raman and Fourier-transform infrared spectroscopy were used to determine the physicochemical and crystallographic properties of type II breast calcifications located in formalin fixed paraffin embedded DCIS breast tissue samples. Multiple calcium phosphate phases were identified across the calcifications, distributed in different patterns. Hydroxyapatite was
the dominant mineral, with magnesium whitlockite found at the calcification edge. Amorphous calcium
phosphate and octacalcium phosphate were also identified close to the calcification edge at the apparent
mineral/matrix barrier. Crystallographic features of hydroxyapatite also varied across the calcifications, with
higher crystallinity centrally, and highest carbonate substitution at the calcification edge. Protein was also
differentially distributed across the calcification and the surrounding soft tissue, with collagen and β-pleated
protein features present to differing extents. Combination of analytical techniques in this study was essential
to understand the heterogeneity of breast calcifications and how this may link crystallographic and physicochemical properties of calcifications to the surrounding tissue microenvironment.
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Mar 2022
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