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Purpose: Proficient DNA repair by homologous recombination (HR) facilitates resistance to chemoradiation in glioma stem cells (GSC). We evaluated whether compromising HR by targeting HSP90, a molecular chaperone required for the function of key HR proteins, using onalespib, a long-acting, brain-penetrant HSP90 inhibitor, would sensitize high-grade gliomas to chemoradiation in vitro and in vivo., Experimental Design: The ability of onalespib to deplete HR client proteins, impair HR repair capacity, and sensitize glioblastoma (GBM) to chemoradiation was evaluated in vitro in GSCs, and in vivo using zebrafish and mouse intracranial glioma xenograft models. The effects of HSP90 inhibition on the transcriptome and cytoplasmic proteins was assessed in GSCs and in ex vivo organotypic human glioma slice cultures., Results: Treatment with onalespib depleted CHK1 and RAD51, two key proteins of the HR pathway, and attenuated HR repair, sensitizing GSCs to the combination of radiation and temozolomide (TMZ). HSP90 inhibition reprogrammed the transcriptome of GSCs and broadly altered expression of cytoplasmic proteins including known and novel client proteins relevant to GSCs. The combination of onalespib with radiation and TMZ extended survival in a zebrafish and a mouse xenograft model of GBM compared with the standard of care (radiation and TMZ) or onalespib with radiation., Conclusions: The results of this study demonstrate that targeting HR by HSP90 inhibition sensitizes GSCs to radiation and chemotherapy and extends survival in zebrafish and mouse intracranial models of GBM. These results provide a preclinical rationale for assessment of HSP90 inhibitors in combination with chemoradiation in patients with GBM., (©2022 American Association for Cancer Research.)

Engineering cysteines at specific sites in antibodies to create well-defined ADCs for the treatment of cancer is a promising approach to increase the therapeutic index and helps to streamline the manufacturing process. Here, we report the development of an in silico screening procedure to select for optimal sites in an antibody to which a hydrophobic linker-drug can be conjugated. Sites were identified inside the cavity that is naturally present in the Fab part of the antibody. Conjugating a linker-drug to these sites demonstrated the ability of the antibody to shield the hydrophobic character of the linker-drug while resulting ADCs maintained their cytotoxic potency in vitro . Comparison of site-specific ADCs versus randomly conjugated ADCs in an in vivo xenograft model revealed improved efficacy and exposure. We also report a selective reducing agent that is able to reduce the engineered cysteines while leaving the interchain disulfides in the oxidized state. This enables us to manufacture site-specific ADCs without introducing impurities associated with the conventional reduction/oxidation procedure for site-specific conjugation.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP ( m -DHP-BDP and m -DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m -DHP-Cy5 is in stark contrast to the cellular uptake of m -DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells., Competing Interests: CJvK and CMT were employees of MSD (Organon)., (This journal is © The Royal Society of Chemistry.)

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide-based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH. In this study, we present the signaling mechanisms of a newly developed LMW dihydropyridine agonist of the FSHR, Org 214444-0. Org 214444-0 is shown to be a stereoselective, nanomolar potent FSHR agonist and selective over the structurally related LHR and TSHR. Org 214444-0 is an allosteric agonist interacting with the transmembrane region of the FSHR. When co-incubated with FSH, Org 214444-0 augments FSH's potency in binding (6.5-fold) and adenylyl cyclase/cAMP activation (3.5-fold) in a concentration-dependent manner. Like FSH, Org 214444-0 induces FSHR internalization and is only marginally effective in stimulating phospholipase C. Moreover, Org 214444-0 stimulates cAMP and estradiol production in human granulosa cells in culture and supports the follicular phase in mature female rats. We conclude that Org 214444-0 is a bonafide FSHR agonist., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Purpose: Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0., Experimental Approach: Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants., Key Results: Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies., Conclusions and Implications: Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO., (© 2011 Merck Sharp & Dohme Corp. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

During recent decades minor innovative drugs have been developed for the female contraceptive market and they all contain steroidal progestagens (and estrogens) that act centrally and have side effects that can be attributed to this central action. In this study, we present an innovative tissue-specific approach for female contraception by low molecular weight (LMW) FSH receptor (FSHR) agonists, which interact with the FSHR that is dominantly expressed in the granulosa cells. The oral administration of LMW FSHR agonists with a short circulation time, induced formation of luteinized unruptured follicles (LUFs) from the Graafian follicles, thereby preventing the release of the oocyte. The short-acting LMW FSHR compounds were fully agonistic to FSHR (EC(50)=4-5 nM). In an isolated mouse follicle culture, a short incubation period (2 h) resulted in inhibition of follicular rupture, where continuous incubation induced follicle growth. Pharmacokinetics after oral administration showed a surge-like exposure in rats and monkeys. Oral administration of short-acting LMW FSHR agonists inhibited ovulation at 10 mg/kg in rats and guinea pigs by generating LUFs without affecting cyclicity. Also, inhibition of follicular rupture was shown to be reversible within one cycle. Finally, LUFs were induced without affecting the hormonal cyclicity in cynomolgus monkeys, a mono-ovulatory species. In healthy women LUF formation occurs naturally, with a LUF acting as corpus luteum that produces enough progesterone to ensure normal menstrual cyclicity. Together with the presented data this indicates that the innovative approach with short-acting LMW FSHR agonists could lead to oral contraception for females at the ovarian level.

Ovarian hyperstimulation syndrome (OHSS) incidentally occurs in controlled ovarian stimulation protocols and is associated with human chorionic gonadotropin (hCG) administration. OHSS is caused by increased vascular permeability (VP) and thought to be mediated by hypersecretion of vascular endothelial growth factor (VEGF) by granulosa cells. Low molecular weight (LMW)-LH agonists have a similar mode of action but a shorter half-life compared with hCG, which could potentially lead to a clinical benefit in reducing the risk for OHSS in controlled ovarian stimulation protocols. The objective of this study is to investigate the role of an orally active LMW-LH agonist in OHSS induction compared with recombinant LH (rec-LH) and hCG. Immature rats were hyperstimulated with pregnant mare serum gonadotropin, and ovulation was induced by hCG, rec-LH or a LMW-LH agonist. The degree of VP was determined by Evans Blue in the abdominal cavity. Ovaries were weighed, and VEGF concentration in the ovary was determined. Pregnant mare serum gonadotropin stimulation followed by single-dose hCG or rec-LH resulted in clear enlargement of the ovaries and increased VP and VEGF levels. However, ovulation induction with a single dose of the LMW-LH agonist did not result in increased VP and VEGF levels, and even multiple dosing to mimic a longer exposure did not induce OHSS symptoms. In conclusion, we demonstrated that the oral LMW-LH agonist did not induce VP in rat, indicative for OHSS, possibly due to reduced VEGF production. If this is translatable to human, this could potentially represent a clinical benefit in reducing the risk for OHSS when using these compounds in controlled ovarian stimulation protocols.

The structural resemblance of the luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) impedes selective agonistic targeting of one of those by low molecular weight (LMW) ligands. In the present study, we describe a series of dimeric ligands consisting of a LMW agonist with dual activity on the FSHR and the LHR linked to a selective FSHR antagonist. Biological evaluation shows these compounds to be potent and selective LHR agonists, since no agonistic activity on the FSHR was observed. Equimolar mixing of the monomeric counterparts did not yield the pharmacological profile observed for the heterodimeric ligands, and FSHR agonism of the monomeric LHR agonist was still observed. The here-described results show that ligands with unique pharmacological profiles can be obtained by dimerizing monomeric molecules with distinct apposite properties.

Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the monomeric counterparts.

A series of homo- and heterodimeric compounds encompassing the follicle-stimulating hormone receptor (FSHR) antagonist (R)-1 and its inactive conformer (S)-1 connected through ethylene glycol spacers of various lengths is described. Evaluation of these compounds reveals that dimeric compounds, with a spacer of sufficient length, bearing two active copies of the antagonist are more potent relative to dimeric compounds in which one of the active pharmacophores is replaced by an inactive conformer. Interestingly, the opposite trend is observed if a short spacer is used, indicating that these compounds may be valuable tools to study FSHR dimerization in greater detail.

Two series of dimeric ligands for a G-protein-coupled receptor were prepared that differ by the interconnecting spacer system. Biological evaluation revealed that both dimeric series exhibit unique biological properties relative to their monomeric counterparts.The luteinizing hormone receptor (LHR), the follicle-stimulating hormone receptor (FSHR), and the thyroid-stimulating hormone receptor (TSHR) belong to the glycoprotein hormone receptor (GpHR) family. A prominent feature of all endogenous glycoprotein ligands is that they share an identical alpha subunit and acquire their selectivity from the unique beta subunit. Recent developments in pro-fertility research have led to the discovery of several low-molecular-weight agonists for the luteinizing hormone/choriogonadotropin receptor that bind to the transmembrane (TM) region of the LHR. Interestingly, some of these agonists are also able to activate the FSHR. Several research groups have shown that ligand dimerization presents a powerful tool to increase the subtype selectivity for structurally related G-protein-coupled receptors. In this work, we applied the dimerization strategy to GpHRs and explored the effect on receptors with closely related TM regions. Two series of dimeric ligands were prepared that differ in the interconnecting spacer system. Biological evaluation revealed that both series exhibit unique selectivity properties for the LHR, originating from either decreased potency or a decreased efficacy toward the FSHR.

Background: In assisted reproductive technology, human chorionic gonadotrophin (hCG) is administered subcutaneously for the induction of oocyte maturation and ovulation. Our efforts to develop orally bioavailable luteinizing hormone (LH) receptor agonists have led to the discovery of Org 43553, a low molecular weight (LMW) LH receptor (LH-R) agonist., Methods: Org 43553 was tested in vitro and in vivo in pre-clinical pharmacological models to demonstrate efficacy and oral availability., Results: Org 43553 is a potent stimulator of the human LH-R in vitro (EC(50) 3.7 nM). In primary mouse Leydig cells, Org 43553 stimulated testosterone production. Pharmacokinetic analyses showed high oral bioavailability in rats (79%) and dogs (44%) with a shorter half-life compared with hCG (3.4 versus 5.6 h in the rat). Ovulation induction by Org 43553 was demonstrated in immature mice as well as in cyclic rats after single-dose oral administration (50 mg/kg). The ovulated oocytes were of good quality as demonstrated by successful fertilization and implantation of normal embryos. In male rats, testosterone production was substantially induced after oral administration., Conclusions: Org 43553 is the first LMW LH-R mimetic with demonstrated in vivo efficacy upon oral administration and could therefore replace subcutaneously administered hCG. The elimination half-life of Org 43553 is substantially shorter than hCG, which could potentially represent a clinical benefit in reducing the risk of ovarian hyperstimulation syndrome (OHSS).

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) activate the LH receptor/cyclic AMP (cAMP) signaling pathway to induce ovulation. As an alternative to parenterally administered hCG to treat anovulatory infertility, orally active low molecular weight (LMW) LHR agonists have been developed at Organon. In this paper, we present the mechanism of action of a prototypic, nanomolar potent and almost full LHR agonist, Org 43553. Org 43553 interacts with the endodomain of the LHR, whereas LH acts via the N-terminal exodomain. LH stimulates the cAMP pathway with an EC50 of 35 pM, but this stimulation is not antagonized by simultaneous incubation with Org 43553. At nanomolar concentrations, LH also stimulates phospholipase C (PLC), but Org 43553 is hardly able to do so. In contrast, Org 43553 inhibits LH-induced PLC (IC50 approximately 10 nM). While Org 43553 stimulates dissociation of [125I]hCG from the LHR and reduces [125I]hCG binding, LH reduces specific [3H]Org 43553 binding. We conclude that Org 43553 is a signaling-selective, allosteric LHR agonist. We hypothesize that Org 43553 and LH induce a similar LHR conformation necessary for activating adenylyl cyclase, which initiates most, if not all, physiological responses of LH.

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.

The luteinizing hormone (LH) receptor plays a pivotal role in reproduction. The high-molecular-weight (HMW) human chorionic gonadotropin (hCG) and LH are the endogenous ligands of this receptor and bind to its large N terminus. The present study characterizes the binding of a new low-molecular-weight (LMW) radioligand, [(3)H]5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-yl-acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-butylamide (Org 43553), at the LH receptor. Equilibrium saturation and displacement assays were developed and optimized. Specific binding of [(3)H]Org 43553 to CHO-K1 cell membranes expressing the human LH receptor and a cAMP response element-luciferase reporter gene was saturable with a K(D) value of 2.4 +/- 0.4 nM and a B(max) value of 1.6 +/- 0.2 pmol/mg protein. Affinities of five LMW analogs of Org 43553 were determined. All displaced the radioligand competitively, with K(i) values ranging from 3.3 to 100 nM. Finally, the potency of these compounds in a cAMP-induced luciferase assay was also determined. There was a high correlation between affinity and potency (r = 0.99; P < 0.0001) of these compounds. In the search for LMW ligands, which bind allosterically to the seven-transmembrane domain of the LH receptor, a HMW radioligand (e.g., (125)I-hCG) is not suitable as it is not displaced by a LMW compound. Therefore, [(3)H]Org 43553, a new radioligand with good binding properties, allows screening for new LMW ligands that mimic the action of the endogenous hormone at the LH receptor.

G protein coupled receptors (GPCRs) are important drug targets in pharmaceutical research. Traditionally, most research efforts have been devoted towards the design of small molecule agonists and antagonists. An interesting, yet poorly investigated class of GPCR modulators comprise the bivalent ligands, in which two receptor pharmacophores are incorporated. Here, we set out to develop a general strategy for the synthesis of bivalent compounds that are projected to bind to the human gonadotropin-releasing hormone receptor (GnRHR). Our results on the dimerisation of a known GnRHR antagonist, with as key step the Huisgen 1,3-cycloaddition, and their ability to bind to and antagonize GnRH-induced GnRHR stimulation, are presented here.

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.

Substituted 6-amino-4-phenyl-tetrahydroquinoline derivatives are described that are antagonists for the G(s)-protein-coupled human follicle-stimulating hormone (FSH) receptor. These compounds show high antagonistic efficacy in vitro using a CHO cell line expressing the human FSH receptor. Antagonist 10 also showed a submicromolar IC(50) in a more physiologically relevant rat granulosa cell assay and was found to significantly inhibit follicle growth and ovulation in an ex vivo mouse model. This compound class may open the way toward a novel, nonsteroidal approach for contraception.