5 research outputs found
Label-Free Bottom-Up Proteomic Workflow for Simultaneously Assessing the Target Specificity of Covalent Drug Candidates and Their Off-Target Reactivity to Selected Proteins
Although
designed covalent inhibitors as drug candidates offer
several unique advantages over conventional reversible inhibitors,
including high potency and the potential for less frequent dosing,
there is a general tendency to avoid the covalent mode of action in
drug discovery programs due to concerns regarding immune-mediated
toxicity that can arise from indiscriminate reactivity with off-target
proteins. Therefore, the ability to assess off-target reactivity relative
to target specificity is desirable for optimizing covalent drug candidates
in the early discovery stage. One concern with current surrogate nucleophile
trapping approaches is that they employ a simplistic model nucleophile
such as glutathione, which may not reliably reflect the covalent interactions
with cellular or extracellular proteins. One way to get a more relevant
reactivity assessment is to directly measure the ability of an inhibitor
to covalently modify nucelophilic amino acids on biologically relevant
proteins, both on- and off-target. In this article, we describe a
label-free bottom-up proteomic workflow for simultaneous evaluation
of target binding and off-target reactivity of covalent drug candidates
to selected proteins at the peptide level. Ibrutinib, a covalent drug
targeting the active site of BTK protein, was used as a model compound
to demonstrate the feasibility of the workflow. The compound was incubated
with a mixture of target protein, Bruton’s tyrosine kinase
(BTK), and two abundant proteins in blood, hemoglobin (Hb) and human
serum albumin (HSA), and then the ibrutinib modification sites were
determined utilizing a bottom-up proteomic approach. A non-BTK specific
model compound (<b>1</b>) known to modify cysteine residues
was also included. By comparing the extent of off-target modifications
to the targeted BTK C481 binding in a wide compound concentration
range, we were able to determine the concentration where maximum target
binding was achieved with minimal off-target reactivity. The generic
label-free bottom-up proteomics workflow described in this article
should be useful in the rank order assessment of off-target reactivity
vs on-target reactivity of covalent drug candidates in the early drug
discovery stage
Label-Free Bottom-Up Proteomic Workflow for Simultaneously Assessing the Target Specificity of Covalent Drug Candidates and Their Off-Target Reactivity to Selected Proteins
Although
designed covalent inhibitors as drug candidates offer
several unique advantages over conventional reversible inhibitors,
including high potency and the potential for less frequent dosing,
there is a general tendency to avoid the covalent mode of action in
drug discovery programs due to concerns regarding immune-mediated
toxicity that can arise from indiscriminate reactivity with off-target
proteins. Therefore, the ability to assess off-target reactivity relative
to target specificity is desirable for optimizing covalent drug candidates
in the early discovery stage. One concern with current surrogate nucleophile
trapping approaches is that they employ a simplistic model nucleophile
such as glutathione, which may not reliably reflect the covalent interactions
with cellular or extracellular proteins. One way to get a more relevant
reactivity assessment is to directly measure the ability of an inhibitor
to covalently modify nucelophilic amino acids on biologically relevant
proteins, both on- and off-target. In this article, we describe a
label-free bottom-up proteomic workflow for simultaneous evaluation
of target binding and off-target reactivity of covalent drug candidates
to selected proteins at the peptide level. Ibrutinib, a covalent drug
targeting the active site of BTK protein, was used as a model compound
to demonstrate the feasibility of the workflow. The compound was incubated
with a mixture of target protein, Bruton’s tyrosine kinase
(BTK), and two abundant proteins in blood, hemoglobin (Hb) and human
serum albumin (HSA), and then the ibrutinib modification sites were
determined utilizing a bottom-up proteomic approach. A non-BTK specific
model compound (<b>1</b>) known to modify cysteine residues
was also included. By comparing the extent of off-target modifications
to the targeted BTK C481 binding in a wide compound concentration
range, we were able to determine the concentration where maximum target
binding was achieved with minimal off-target reactivity. The generic
label-free bottom-up proteomics workflow described in this article
should be useful in the rank order assessment of off-target reactivity
vs on-target reactivity of covalent drug candidates in the early drug
discovery stage
Identification of Human Liver Microsomal Proteins Adducted by a Reactive Metabolite Using Shotgun Proteomics
Covalent modification of cellular
proteins by chemically reactive
compounds/metabolites has the potential to disrupt biological function
and elicit serious adverse drug reactions. Information on the nature
and binding patterns of protein targets are critical toward understanding
the mechanism of drug induced toxicity. Protein covalent binding studies
established in liver microsomes can quantitively estimate the extent
of protein modification, but they provide little information on the
nature of the modified proteins. In this article, we describe a label-free
shotgun proteomic workflow for the identification of target proteins
modified in situ by reactive metabolites in human liver microsome
incubations. First, we developed a shotgun proteomic workflow for
the characterization of the human liver microsomal subproteome, which
consists of predominately membrane-bound proteins. Human liver microsomes
were solubilized with a combination of MS-compatible organic solvents
followed by protein reduction, alkylation, and tryptic digestion.
The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins
were identified by database searching. This workflow led to the successful
identification of 329 human liver microsomal subproteome proteins
with 1% FDR (false discovery rate). The same method was then applied
to identify the modifications of human liver microsomal proteins by
a known reactive metabolite 2-(methylsulfonyl)benzo[<i>d</i>]thiazole (<b>2</b>), either after incubation directly with <b>2</b> or with its parent compound 2-(methylthio)benzo[<i>d</i>]thiazole (<b>1</b>). A total of 19 modified constituent
peptides which could be mapped to 18 proteins were identified in human
liver microsomes incubated directly with <b>2</b>. Among these,
5 modified constituent peptides which could be mapped to 4 proteins
were identified in incubation with <b>1</b>, which is known
to generate <b>2</b> in human liver microsomal incubations.
This label-free workflow is generally applicable to the identification
and characterization of proteins adducted with reactive metabolites
in complex matrices and may serve as a valuable tool to understand
the link between protein targets and clinically relevant toxicities
Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric Inhibitor (BMS-929075) Advanced to Phase 1 Clinical Studies
The hepatitis C virus (HCV) NS5B
replicase is a prime target for
the development of direct-acting antiviral drugs for the treatment
of chronic HCV infection. Inspired by the overlay of bound structures
of three structurally distinct NS5B palm site allosteric inhibitors,
the high-throughput screening hit anthranilic acid <b>4</b>,
the known benzofuran analogue <b>5</b>, and the benzothiadiazine
derivative <b>6</b>, an optimization process utilizing the simple
benzofuran template <b>7</b> as a starting point for a fragment
growing approach was pursued. A delicate balance of molecular properties
achieved via disciplined lipophilicity changes was essential to achieve
both high affinity binding and a stringent targeted absorption, distribution,
metabolism, and excretion profile. These efforts led to the discovery
of BMS-929075 (<b>37</b>), which maintained ligand efficiency
relative to early leads, demonstrated efficacy in a triple combination
regimen in HCV replicon cells, and exhibited consistently high oral
bioavailability and pharmacokinetic parameters across preclinical
animal species. The human PK properties from the Phase I clinical
studies of <b>37</b> were better than anticipated and suggest
promising potential for QD administration
Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists
G protein-coupled
receptor 40 (GPR40) has become an attractive
target for the treatment of diabetes since it was shown clinically
to promote glucose-stimulated insulin secretion. Herein, we report
our efforts to develop highly selective and potent GPR40 agonists
with a dual mechanism of action, promoting both glucose-dependent
insulin and incretin secretion. Employing strategies to increase polarity
and the ratio of sp<sup>3</sup>/sp<sup>2</sup> character of the chemotype,
we identified BMS-986118 (compound <b>4</b>), which showed potent
and selective GPR40 agonist activity <i>in vitro</i>. <i>In vivo</i>, compound <b>4</b> demonstrated insulinotropic
efficacy and GLP-1 secretory effects resulting in improved glucose
control in acute animal models