Kevin Madauss - Academia.edu (original) (raw)

Papers by Kevin Madauss

Research paper thumbnail of Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Clinical Cancer Research

Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the deve... more Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. Experimental Design: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1-and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dualtumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

Research paper thumbnail of FXR agonist activity of conformationally constrained analogs of GW 4064

Bioorganic Medicinal Chemistry Letters, Aug 15, 2009

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Re... more Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Research paper thumbnail of Mutations in the mineralocorticoid receptor ligand binding domain polypeptide that permit structural determination of low affinity ligand complexes and screening methods employing same

Research paper thumbnail of Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

Bioorganic Medicinal Chemistry Letters, Aug 15, 2009

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bo... more Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Research paper thumbnail of Structure of neurolysin reveals a deep channel that limits substrate access

Proceedings of the National Academy of Sciences, 2001

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural e... more The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded ␤-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Research paper thumbnail of Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

Proceedings of the National Academy of Sciences, 2009

APPLIED BIOLOGICAL SCIENCES CHEMISTRY Fig. 3. (A) TR (NF-B, open circles) and TA (MMTV, closed ci... more APPLIED BIOLOGICAL SCIENCES CHEMISTRY Fig. 3. (A) TR (NF-B, open circles) and TA (MMTV, closed circles) dose responses for compound 11 and (B) for compound 12. (C) Peptide binding profiles for compounds 11 (hatched bars) and 12 (checked bars) compared with fluticasone propionate (black bars) and mifepristone (RU-486) (white bars).

Research paper thumbnail of Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group

Nature Chemical Biology, 2013

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the m... more In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Research paper thumbnail of A Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor Modulator

Molecular Endocrinology, 2007

One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and ... more One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P 4 ). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the co-activators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P 4induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P 4 -like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P 4 -like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P 4 , and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus. (Molecular Endocrinology 21: [1066][1067][1068][1069][1070][1071][1072][1073][1074][1075][1076][1077][1078][1079][1080][1081] 2007)

Research paper thumbnail of The evolution of progesterone receptor ligands

Medicinal Research Reviews, 2007

Progesterone is one of the first nuclear receptor hormones to be described functionally and subse... more Progesterone is one of the first nuclear receptor hormones to be described functionally and subsequently approached as a drug target. Because progesterone (1) affects both menstruation and gestation via the progesterone receptor (PR), research aimed at modulating its activity is usually surrounded by controversy. However, ligands for PR were developed into drugs, and their evolution can be crudely divided into three periods: (1) drug-like steroids that mimic the gestational properties of progesterone; (2) drug-like steroids with different properties from progesterone and expanded therapeutic applications; and (3) non-steroidal PR ligands with improved selectivity and modulator properties and further expanded therapeutic applications. Although the latter have yet to see widespread clinical applications, their development is founded on a half century of research, and they represent the future for this drug target.

Research paper thumbnail of Progesterone Receptor Ligand Binding Pocket Flexibility:  Crystal Structures of the Norethindrone and Mometasone Furoate Complexes

Journal of Medicinal Chemistry, 2004

Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom ... more Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom at the 17alpha position, other potent steroid agonists such as norethindrone and mometasone furoate have larger substituents at this position that are accommodated by the PR ligand binding pocket. Crystallographic analysis of PR ligand binding domain complexes clearly demonstrated that these moieties were accommodated by local shifts of the protein main chain and by adoption of alternative side chain rotamer conformations of ligand-proximal amino acids. These conformational changes imparted a ligand-specific volume to the binding pocket, from 490 A3 in the metribolone complex to 520 A3 in the norethindrone complex, 565 A3 in the progesterone complex, and 730 A3 in the mometasone furoate complex. Despite these marked alterations in binding pocket volume, critical interactions essential for establishment of an active AF2 conformation were maintained.

Research paper thumbnail of A Ligand-mediated Hydrogen Bond Network Required for the Activation of the Mineralocorticoid Receptor

Journal of Biological Chemistry, 2005

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activit... more Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn 770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr 945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.

Research paper thumbnail of Generation and Characterization of Human Steroidogenic Factor 1 LBD Crystals with and without Bound Cofactor Peptide

Endocrine Research, 2004

The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the... more The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the adrenal gland and gonads, and in sexual differentiation. SF1 performs this pivotal function through the regulation of hormone expression that is essential for organogenesis and endocrine homeostasis. SF1 is a member of a nuclear receptor subclass that contains LRH1 and the Drosophila receptor FTZ-F1. To date, a natural ligand has not been reported for any member of this subfamily. Here we report the crystallization and characterization of the ligand-binding domain (LBD) of human SF1 from two different crystal forms: a binary complex with fortuitous ligand and a ternary complex with the same ligand and a peptide containing a motif of a nuclear receptor cofactor. The structural determination of the binary complex required the use of sulfur SAD phasing, a relatively new technique that uses anomalous diffraction from the endogenous sulfur atoms present in the protein. The structure of the ternary complex was determined by multiple wavelength anomalous diffraction (MAD) using seleno-methionine substituted SF1. Preliminary analysis suggested SF1 contained a fortuitous ligand in the binding pocket. This ligand may account for the relatively high basal activity observed for SF1 in cofactor recruitment and cell-based assays.

Research paper thumbnail of Structural Analyses Reveal Phosphatidyl Inositols as Ligands for the NR5 Orphan Receptors SF-1 and LRH-1

Research paper thumbnail of The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

Bioorganic & Medicinal Chemistry Letters, 2008

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-... more The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

Research paper thumbnail of FXR agonist activity of conformationally constrained analogs of GW 4064

Bioorganic & Medicinal Chemistry Letters, 2009

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Re... more Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Research paper thumbnail of Discovery of non-steroidal mifepristone mimetics: Pyrazoline-based PR antagonists

Bioorganic & Medicinal Chemistry Letters, 2005

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and ... more Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.

Research paper thumbnail of Substituted isoxazole analogs of farnesoid X receptor (FXR) agonist GW4064

Bioorganic & Medicinal Chemistry Letters, 2009

Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazole... more Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.

Research paper thumbnail of Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

Bioorganic & Medicinal Chemistry Letters, 2009

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bo... more Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Research paper thumbnail of Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP)

Bioorganic & Medicinal Chemistry Letters, 2008

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyr... more We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Research paper thumbnail of Conformationally constrained farnesoid X receptor (FXR) agonists: Alternative replacements of the stilbene

Bioorganic & Medicinal Chemistry Letters, 2011

To further explore the optimum placement of the acid moiety in conformationally constrained analo... more To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.

Research paper thumbnail of Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Clinical Cancer Research

Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the deve... more Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. Experimental Design: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1-and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dualtumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

Research paper thumbnail of FXR agonist activity of conformationally constrained analogs of GW 4064

Bioorganic Medicinal Chemistry Letters, Aug 15, 2009

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Re... more Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Research paper thumbnail of Mutations in the mineralocorticoid receptor ligand binding domain polypeptide that permit structural determination of low affinity ligand complexes and screening methods employing same

Research paper thumbnail of Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

Bioorganic Medicinal Chemistry Letters, Aug 15, 2009

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bo... more Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Research paper thumbnail of Structure of neurolysin reveals a deep channel that limits substrate access

Proceedings of the National Academy of Sciences, 2001

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural e... more The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded ␤-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Research paper thumbnail of Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

Proceedings of the National Academy of Sciences, 2009

APPLIED BIOLOGICAL SCIENCES CHEMISTRY Fig. 3. (A) TR (NF-B, open circles) and TA (MMTV, closed ci... more APPLIED BIOLOGICAL SCIENCES CHEMISTRY Fig. 3. (A) TR (NF-B, open circles) and TA (MMTV, closed circles) dose responses for compound 11 and (B) for compound 12. (C) Peptide binding profiles for compounds 11 (hatched bars) and 12 (checked bars) compared with fluticasone propionate (black bars) and mifepristone (RU-486) (white bars).

Research paper thumbnail of Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group

Nature Chemical Biology, 2013

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the m... more In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Research paper thumbnail of A Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor Modulator

Molecular Endocrinology, 2007

One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and ... more One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P 4 ). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the co-activators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P 4induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P 4 -like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P 4 -like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P 4 , and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus. (Molecular Endocrinology 21: [1066][1067][1068][1069][1070][1071][1072][1073][1074][1075][1076][1077][1078][1079][1080][1081] 2007)

Research paper thumbnail of The evolution of progesterone receptor ligands

Medicinal Research Reviews, 2007

Progesterone is one of the first nuclear receptor hormones to be described functionally and subse... more Progesterone is one of the first nuclear receptor hormones to be described functionally and subsequently approached as a drug target. Because progesterone (1) affects both menstruation and gestation via the progesterone receptor (PR), research aimed at modulating its activity is usually surrounded by controversy. However, ligands for PR were developed into drugs, and their evolution can be crudely divided into three periods: (1) drug-like steroids that mimic the gestational properties of progesterone; (2) drug-like steroids with different properties from progesterone and expanded therapeutic applications; and (3) non-steroidal PR ligands with improved selectivity and modulator properties and further expanded therapeutic applications. Although the latter have yet to see widespread clinical applications, their development is founded on a half century of research, and they represent the future for this drug target.

Research paper thumbnail of Progesterone Receptor Ligand Binding Pocket Flexibility:  Crystal Structures of the Norethindrone and Mometasone Furoate Complexes

Journal of Medicinal Chemistry, 2004

Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom ... more Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom at the 17alpha position, other potent steroid agonists such as norethindrone and mometasone furoate have larger substituents at this position that are accommodated by the PR ligand binding pocket. Crystallographic analysis of PR ligand binding domain complexes clearly demonstrated that these moieties were accommodated by local shifts of the protein main chain and by adoption of alternative side chain rotamer conformations of ligand-proximal amino acids. These conformational changes imparted a ligand-specific volume to the binding pocket, from 490 A3 in the metribolone complex to 520 A3 in the norethindrone complex, 565 A3 in the progesterone complex, and 730 A3 in the mometasone furoate complex. Despite these marked alterations in binding pocket volume, critical interactions essential for establishment of an active AF2 conformation were maintained.

Research paper thumbnail of A Ligand-mediated Hydrogen Bond Network Required for the Activation of the Mineralocorticoid Receptor

Journal of Biological Chemistry, 2005

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activit... more Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn 770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr 945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.

Research paper thumbnail of Generation and Characterization of Human Steroidogenic Factor 1 LBD Crystals with and without Bound Cofactor Peptide

Endocrine Research, 2004

The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the... more The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the adrenal gland and gonads, and in sexual differentiation. SF1 performs this pivotal function through the regulation of hormone expression that is essential for organogenesis and endocrine homeostasis. SF1 is a member of a nuclear receptor subclass that contains LRH1 and the Drosophila receptor FTZ-F1. To date, a natural ligand has not been reported for any member of this subfamily. Here we report the crystallization and characterization of the ligand-binding domain (LBD) of human SF1 from two different crystal forms: a binary complex with fortuitous ligand and a ternary complex with the same ligand and a peptide containing a motif of a nuclear receptor cofactor. The structural determination of the binary complex required the use of sulfur SAD phasing, a relatively new technique that uses anomalous diffraction from the endogenous sulfur atoms present in the protein. The structure of the ternary complex was determined by multiple wavelength anomalous diffraction (MAD) using seleno-methionine substituted SF1. Preliminary analysis suggested SF1 contained a fortuitous ligand in the binding pocket. This ligand may account for the relatively high basal activity observed for SF1 in cofactor recruitment and cell-based assays.

Research paper thumbnail of Structural Analyses Reveal Phosphatidyl Inositols as Ligands for the NR5 Orphan Receptors SF-1 and LRH-1

Research paper thumbnail of The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

Bioorganic & Medicinal Chemistry Letters, 2008

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-... more The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

Research paper thumbnail of FXR agonist activity of conformationally constrained analogs of GW 4064

Bioorganic & Medicinal Chemistry Letters, 2009

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Re... more Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Research paper thumbnail of Discovery of non-steroidal mifepristone mimetics: Pyrazoline-based PR antagonists

Bioorganic & Medicinal Chemistry Letters, 2005

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and ... more Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.

Research paper thumbnail of Substituted isoxazole analogs of farnesoid X receptor (FXR) agonist GW4064

Bioorganic & Medicinal Chemistry Letters, 2009

Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazole... more Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.

Research paper thumbnail of Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

Bioorganic & Medicinal Chemistry Letters, 2009

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bo... more Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Research paper thumbnail of Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP)

Bioorganic & Medicinal Chemistry Letters, 2008

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyr... more We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Research paper thumbnail of Conformationally constrained farnesoid X receptor (FXR) agonists: Alternative replacements of the stilbene

Bioorganic & Medicinal Chemistry Letters, 2011

To further explore the optimum placement of the acid moiety in conformationally constrained analo... more To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.