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1.
ACS Chem Biol ; 16(9): 1757-1769, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34406751

ABSTRACT

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.


Subject(s)
Complementarity Determining Regions/chemistry , Immunoglobulin Fragments/chemistry , Peptides, Cyclic/chemical synthesis , Amino Acid Sequence , Animals , Cattle , Immunoglobulin Fragments/blood , Immunoglobulin Fragments/pharmacology , Male , Models, Molecular , Peptides, Cyclic/blood , Peptides, Cyclic/pharmacokinetics , Protein Binding , Protein Domains , Protein Folding , Rats, Sprague-Dawley , Solid-Phase Synthesis Techniques , Tandem Mass Spectrometry , Thermodynamics
2.
J Med Chem ; 61(15): 6705-6723, 2018 08 09.
Article in English | MEDLINE | ID: mdl-29952567

ABSTRACT

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.


Subject(s)
Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/pharmacokinetics , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Piperazines/pharmacology , Piperazines/pharmacokinetics , Piperidines/pharmacology , Transplantation, Homologous , Administration, Oral , Animals , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacokinetics , Immunosuppressive Agents/pharmacology , Mice , Molecular Docking Simulation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Piperazines/administration & dosage , Piperazines/metabolism , Piperidines/administration & dosage , Piperidines/metabolism , Protein Conformation
3.
J Biol Chem ; 287(32): 26464-77, 2012 Aug 03.
Article in English | MEDLINE | ID: mdl-22696217

ABSTRACT

LRP5 and LRP6 are proteins predicted to contain four six-bladed ß-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-5/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Wnt1 Protein/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Bone Morphogenetic Proteins/genetics , Calorimetry , Cell Line , Crystallography , DNA, Complementary , Genetic Markers/genetics , Humans , Low Density Lipoprotein Receptor-Related Protein-5/genetics , Low Density Lipoprotein Receptor-Related Protein-6/chemistry , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Wnt1 Protein/genetics
5.
Bioorg Med Chem Lett ; 19(2): 397-400, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19059779

ABSTRACT

A series of quinoxaline inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and optimisation of the series was performed on the basis of this structure. Future directions for development of the series are discussed together with the identification of a novel quinoline scaffold.


Subject(s)
Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Quinoxalines/pharmacology , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemistry , Quinoxalines/chemistry , Structure-Activity Relationship
6.
J Med Chem ; 51(24): 8124-34, 2008 Dec 25.
Article in English | MEDLINE | ID: mdl-19053774

ABSTRACT

Vasopressin (AVP) is a hormone that stimulates an increase in water permeability through activation of V2 receptors in the kidney. The analogue of AVP, desmopressin, has proven an effective drug for diseases where a reduction of urine output is desired. However, its peptidic nature limits its bioavailability. We report herein the discovery of potent, nonpeptidic, benzylurea derived agonists of the vasopressin V2 receptor. We describe substitutions on the benzyl group to give improvements in potency and subsequent modifications to the urea end group to provide improvements in solubility and increased oral efficacy in a rat model of diuresis. The lead compound 20e (VA106483) is reported for the first time and has been selected for clinical development.


Subject(s)
Chemistry, Pharmaceutical/methods , Receptors, Vasopressin/agonists , Urea/chemistry , Administration, Oral , Animals , Caco-2 Cells , Diuresis , Dose-Response Relationship, Drug , Drug Design , Drug Evaluation, Preclinical , Humans , Models, Chemical , Rats , Rats, Brattleboro , Solubility
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