18 research outputs found
Development of a small molecule that corrects misfolding and increases secretion of Z α1 -antitrypsin.
Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency
Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening
AbstractThe identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.</jats:p
Predicting Electrophoretic Mobility of Protein–Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules
Selection
of target-binding ligands from DNA-encoded libraries
of small molecules (DELSMs) is a rapidly developing approach in drug-lead
discovery. Methods of kinetic capillary electrophoresis (KCE) may
facilitate highly efficient homogeneous selection of ligands from
DELSMs. However, KCE methods require accurate prediction of electrophoretic
mobilities of protein–ligand complexes. Such prediction, in
turn, requires a theory that would be applicable to DNA tags of different
structures used in different DELSMs. Here we present such a theory.
It utilizes a model of a globular protein connected, through a single
point (small molecule), to a linear DNA tag containing a combination
of alternating double-stranded and single-stranded DNA (dsDNA and
ssDNA) regions of varying lengths. The theory links the unknown electrophoretic
mobility of protein–DNA complex with experimentally determined
electrophoretic mobilities of the protein and DNA. Mobility prediction
was initially tested by using a protein interacting with 18 ligands
of various combinations of dsDNA and ssDNA regions, which mimicked
different DELSMs. For all studied ligands, deviation of the predicted
mobility from the experimentally determined value was within 11%.
Finally, the prediction was tested for two proteins and two ligands
with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline.
Deviation between the predicted and experimentally determined mobilities
did not exceed 5%. These results confirm the accuracy and robustness
of our model, which makes KCE methods one step closer to their practical
use in selection of drug leads, and diagnostic probes from DELSMs
Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and in vivo activity
The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases
Orally Active Fumagillin Analogues: Transformations of a Reactive Warhead in the Gastric Environment
Semisynthetic
analogues of fumagillin, <b>1</b>, inhibit methionine aminopeptidase-2
(MetAP2) and have entered the clinic for the treatment of cancer.
An optimized fumagillin analogue, <b>3</b> (PPI-2458), was found
to be orally active, despite containing a spiroepoxide function that
formed a covalent linkage to the target protein. In aqueous acid, <b>3</b> underwent ring-opening addition of water and HCl, leading
to four products, <b>4–7</b>, which were characterized
in detail. The chlorohydrin, but not the diol, products inhibited
MetAP2 under weakly basic conditions, suggesting reversion to epoxide
as a step in the mechanism. In agreement, chlorohydrin <b>6</b> was shown to revert rapidly to <b>3</b> in rat plasma. In
an ex vivo assay, rats treated with purified acid degradants demonstrated
inhibition of MetAP2 that correlated with the biochemical activity
of the compounds. Taken together, the results indicate that degradation
of the parent compound was compensated by the formation of active
equivalents leading to a pharmacologically useful level of MetAP2
inhibition
<i>In vivo</i> half-life extension of BMP1/TLL metalloproteinase inhibitors using small-molecule human serum albumin binders
Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer half-lives are highly desirable. One of the most promising approaches to extend the half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of , a small-molecule noncovalent HSA binder, to extend the half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of with BMP1/TLL inhibitors were prepared. In particular, showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies
Development of a small molecule that corrects misfolding and increases secretion of Z α1‐antitrypsin
Abstract Severe α1‐antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1‐antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA‐encoded chemical library to undertake a high‐throughput screen to identify small molecules that bind to, and stabilise Z α1‐antitrypsin. The lead compound blocks Z α1‐antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1‐antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1‐antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that “mutation ameliorating” small molecules can block the aberrant polymerisation that underlies Z α1‐antitrypsin deficiency
Discovery, SAR, and X‑ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library
As a potential target for obesity,
human BCATm was screened against
more than 14 billion DNA encoded compounds of distinct scaffolds followed
by off-DNA synthesis and activity confirmation. As a consequence,
several series of BCATm inhibitors were discovered. One representative
compound (<i>R</i>)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-<i>N</i>-methyl-2′-(methylsulfonamido)-[1,1′-biphenyl]-4-carboxamide
(<b>15e</b>) from a novel compound library synthesized via on-DNA
Suzuki–Miyaura cross-coupling showed BCATm inhibitory activity
with IC<sub>50</sub> = 2.0 μM. A protein crystal structure of <b>15e</b> revealed that it binds to BCATm within the catalytic site
adjacent to the PLP cofactor. The identification of this novel inhibitor
series plus the establishment of a BCATm protein structure provided
a good starting point for future structure-based discovery of BCATm
inhibitors