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Supplementary Table 1

Systemic inhibition of Notch with γ-secretase inhibitors (GSI) decreases multiple myeloma tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine multiple myeloma, treatment with BT-GSI decreased tumor burden and prevented the progression of multiple myeloma-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-resorptive properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of multiple myeloma and associated bone disease.Significance:Development of a bone-targeted Notch inhibitor reduces multiple myeloma growth and mitigates cancer-induced bone destruction without inducing the gastrointestinal toxicity typically associated with inhibition of Notch.

Supplementary figures 1, 2, and 3

Systemic inhibition of Notch with γ-secretase inhibitors (GSI) decreases multiple myeloma tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine multiple myeloma, treatment with BT-GSI decreased tumor burden and prevented the progression of multiple myeloma-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-resorptive properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of multiple myeloma and associated bone disease. Significance: Development of a bone-targeted Notch inhibitor reduces multiple myeloma growth and mitigates cancer-induced bone destruction without inducing the gastrointestinal toxicity typically associated with inhibition of Notch.

Parathyroid hormone (PTH) signaling downstream of the PTH 1 receptor (Pth1r) results in both bone anabolic and catabolic actions by mechanisms not yet fully understood. In this study, we show that Pth1r signaling upregulates the expression of several components of the Notch pathway and that Notch signals contribute to the catabolic actions of PTH in bone. We found that constitutive genetic activation of PTH receptor signaling in osteocytes (caPth1r(Ot)) or treatment with PTH daily increased the expression of several Notch ligands/receptors in bone. In contrast, sustained elevation of endogenous PTH did not change Notch components expression. Deletion of the PTH receptor or Sclerostin overexpression in osteocytes abolished Notch increases by PTH. Further, deleting the canonical Notch transcription factor Rbpjk in osteocytes decreased bone mass and increased resorption and Rankl expression in caPth1r(Ot) mice. Moreover, pharmacological bone-targeted Notch inhibition potentiated the bone mass gain induced by intermittent PTH by reducing bone resorption and preserving bone formation. Thus, Notch activation lies downstream of anabolic signaling driven by PTH actions in osteocytes, and Notch pharmacological inhibition maximizes the bone anabolic effects of PTH.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bortezomib (Btz) is a proteasome inhibitor approved by the FDA to treat multiple myeloma. It also increases bone volume by promoting osteoblast differentiation and inhibiting osteoclastogenesis in mice. However, Btz has severe systemic adverse effects, which would limit its use as a bone anabolic agent. Here, we designed and synthesized a bone-targeted form of Btz by conjugating it to a bisphosphonate (BP) with no antiresorptive activity. We report that BP-Btz inhibited osteoclast formation and bone resorption and stimulated osteoblast differentiation in vitro similar to Btz. In vivo, BP-Btz increased bone volume more effectively than Btz in three mouse models: untreated wild-type mice, mice with ovariectomy, and aged mice with tibial factures. Importantly, BP-Btz had significantly less systemic side effects than Btz, including less thymic cell death, sympathetic nerve damage, and thrombocytopenia, and it improved survival rates in aged mice. Thus, BP-Btz represents a novel anabolic agent to treat conditions, such as postmenopausal and age-related bone loss. Bone targeting is an attractive approach to repurpose approved drugs to treat skeletal diseases. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

Advances in the design of potential bone-selective drugs for the treatment of various bone-related diseases are creating exciting new directions for multiple unmet medical needs. For bone-related cancers, off-target/non-bone toxicities with current drugs represent a significant barrier to the quality of life of affected patients. For bone infections and osteomyelitis, bacterial biofilms on infected bones limit the efficacy of antibiotics because it is hard to access the bacteria with current approaches. Promising new experimental approaches to therapy, based on bone-targeting of drugs, have been used in animal models of these conditions and demonstrate improved efficacy and safety. The success of these drug-design strategies bodes well for the development of therapies with improved efficacy for the treatment of diseases affecting the skeleton. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

The skeleton is affected by numerous primary and metastatic solid and hematopoietic malignant tumors, which can cause localized sites of osteolysis or osteosclerosis that can weaken bones and increase the risk of fractures in affected patients. Chemotherapeutic drugs can eliminate some tumors in bones or reduce their volume and skeletal-related events, but adverse effects on non-target organs can significantly limit the amount of drug that can be administered to patients. In these circumstances, it may be impossible to deliver therapeutic drug concentrations to tumor sites in bones. One attractive mechanism to approach this challenge is to conjugate drugs to bisphosphonates, which can target them to bone where they can be released at diseased sites. Multiple attempts have been made to do this since the 1990s with limited degrees of success. Here, we review the results of pre-clinical and clinical studies made to target FDA-approved drugs and other antineoplastic small molecules to bone to treat diseases affecting the skeleton, including osteoporosis, metastatic bone disease, multiple myeloma and osteosarcoma. Results to date are encouraging and indicate that drug efficacy can be increased and side effects reduced using these approaches. Despite these successes, challenges remain: no drugs have gone beyond small phase 2 clinical trials, and major pharmaceutical companies have shown little interest in the approach to repurpose any of their drugs or to embrace the technology. Nevertheless, interest shown by smaller biotechnology companies in the technology suggests that bone-targeting of drugs with bisphosphonates has a viable future.

Multiple myeloma (MM) is the most common cancer affecting the bone and bone marrow and remains incurable for most patients, novel therapies are therefore needed. Bortezomib (Btz) is an FDA-approved drug for the treatment of patients with MM. However, its severe side effects require a dose reduction or the potential discontinuation of treatment. To overcome this limitation, we conjugated Btz to a bisphosphonate (BP) residue lacking anti-osteoclastic activity using a novel chemical linker and generated a new bone-targeted Btz-based (BP-Btz) proteasome inhibitor. We demonstrated that BP-Btz, but not Btz, bound to bone slices and inhibited the growth of MM cells in vitro. In a mouse model of MM, BP-Btz more effectively reduced tumor burden and bone loss with less systemic side effects than Btz. Thus, BP-Btz may represent a novel therapeutic approach to treat patients with MM.

Further studies of the direct enantioselective α-hydroxymethylation of aldehydes employing the α,α-diarylprolinol trimethylsilyl ether class of organocatalysts are described. This process has proven efficient for access to β-hydroxycarboxylic acids and δ-hydroxy-α,β-unsaturated esters from aldehydes in generally good yields, excellent enantioselectivity, and compatibility with a broad range of functional groups in the aldehyde. The goal of these studies was to identify the critical reaction variables that influence the yield and enantioselectivity of the α-hydroxymethylation process such as catalyst structure, pH of the medium, purity of the reactants and reagents particularly with respect to the presence of acidic impurities, and the nature of the buffer, along with the standard variables including solvent, time, temperature and mixing efficiency. The previously identified intermediate lactol has been further characterized and its reactivity examined. These studies have led to identification of the most critical variables translating directly into improved substrate scope, reproducibility, enantioselectivity, and yields.

Communication between myeloma (MM) cells and cells of the bone marrow via Notch signaling promotes tumor growth/survival and stimulates bone resorption. Systemic inhibition of Notch, using γ-secretase inhibitors (GSIs), decreases MM growth and reduces bone destruction, but the clinical use of GSIs is limited due to dose-limiting severe gut toxicity. To circumvent GSI side effects, we generated a bone specific Notch inhibitor (BT-GSI) by conjugating GSI-XII to a targeting molecule (BT) with high bone affinity using an acid hydrolyzable linker. In vitro, BT-GSI was inactive unless pre-incubated at low pH, and exhibited equal inhibition of Notch target genes in MM cells as unconjugated GSI. Ex vivo, BT-GSI decreased Notch expression and reduced MM growth in bone organ cultures that reproduce acidic conditions in the MM-bone microenvironment. In vivo, treatment with BT-GSI (5mg/kg/3x/wk, i.p.) for 2 wks decreased Notch signaling in bone more efficiently than unconjugated GSI (10mg/kg/5x/wk, i.p.) in naïve mice. In addition, BT-GSI increased cancellous bone mass (30%) and decreased bone resorption by 40%, without affecting bone formation. In contrast, these parameters remained unchanged by GSI. Next, we examined in vivo the impact of BT-GSI on MM growth and bone disease in a preclinical model of established MM. 8-wk-old immunodeficient mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline. hMM injected mice exhibited detectable serum levels of the tumor biomarker human K-light chain (40 ng/mL) and visible osteolytic disease (osteolytic area 1.7 mm2) 3 wks after hMM inoculation. Then, hMM-injected mice were randomized based on tumor levels to two subgroups to receive either BT-GSI (10mg/kg/3x/wk) or vehicle (DMSO) for 3 wks. Saline-injected mice received vehicle injections. BT-GSI selectively decreased Notch gene expression in bone, but had no effect in the brain or gut. Further, BT-GSI did not increase the expression of Adipsin in the gut, a biomarker of gut toxicity, nor showed evidence of gut toxicity at necropsy. Mice treated with BT-GSI exhibited a 45% decrease in tumor burden (168 vs 254 ng/mL human K-light chain) and 50% less osteolytic area compared to vehicle treated mice bearing hMM (4.4 vs 10.2 mm2). Moreover, BT-GSI decreased serum CTX by 30%, but did not affect serum P1NP. Importantly, equimolar administration of the unconjugated BT molecule did not alter MM growth nor prevented bone loss in mice with established MM. In conclusion, these results show that bone-targeted Notch inhibition reduces MM growth and preserves bone mass in mice with established MM. Because BT-GSI shows bone specific Notch inhibition and lacks gut toxicity, it should circumvent the deleterious side effects that limit GSI use in patients. Thus, BT-GSI is a promising approach to inhibit MM growth and to prevent bone loss in MM patients. Citation Format: Adam J. Ferrari, Kevin McAndrews, Jessica H. Nelson, James T. Bell, Venkatesan Srinivasan, Frank H. Ebetino, Robert K. Boeckman Jr, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Disruption of Notch Signaling targeted to the myeloma bone marrow microenvironment simultaneously inhibits tumor growth and prevents bone loss without inducing gut toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 103.

A full account of studies that culminated in the total synthesis of both antipodes and the assignment of its absolute configuration of Saudin, a hypoglycemic natural product. Two approaches are described, the first proceeding though bicyclic lactone intermediates and related second monocyclic esters. The former was obtained via asymmetric Diels–Alder cycloaddition and the latter by an asymmetric annulation protocol. Both approaches employ a Lewis acid promoted Claisen rearrangement, with the successful approach taking advantage of bidentate chelation to control the facial selectivity of the key Claisen rearrangement.

Camphor-derived lactams and other related chiral controllers have been found to react with the Bestmann ylide (triphenylphosphoranylideneketene) upon heating in toluene. The resulting parent ylides provide convenient access to a structurally diverse set of chiral stabilized ylides via functionalization. The utility of these chiral ylides for Wittig reactions has been briefly investigated and the effects of alpha-substitution noted.

A tandem cyclopropyliminium ion rearrangement-Hofmann elimination affords rapid access to 3-vinyl Δ2-pyrrolines in some cases bearing an unsaturated tether in good yield. The resulting dienamines subsequently undergo both inter- and intramolecular [4+2] cycloaddition reactions. Good yields were obtained for a number of cases of intramolecular intermolecular cycloaddition reactions affording cycloadducts in good yields with sufficiently reactive dienophiles such as fumarate, acrylate and methacrolein. Evidence suggests a non-concerted pathway for these cycloaddition reactions.

Several cases of significant enhancement in the diastereoselectivity of acyclic Claisen rearrangement of cyclohexenyl allyl ethers governed principally by remote asymmetric center(s) have been observed when Lewis acid catalysts are employed rather than the usual thermal rearrangement conditions.

Two camphor derived lactams 1 and 2 have been examined as to their utility as chiral auxiliaries for use in asymmetric aldol reactions. The results show good to excellent selectivities can be achieved with 2 in most cases employing diethyl boron enolates. These enolates proved less reactive than anticipated and exhibited a sensitivity to the steric size of the ligands on boron. A model which rationalizes the sense of diastereoselection is presented.

An efficient enantioselective route to the octahydronaphthalene unit present in tetronolide ( 1 ), the stereochemically complex aglycone common to the tetrocarcins, a novel group of antitumor substances, is described. The sequence employs the intramolecular Diels-Alder reaction to control the relative stereochemistry present on the trans decalin ring system, and incorporates a masked acylating agent which should permit coupling of the two key fragments 2 and 3 as demonstrated by reaction of pentaene 4 with methanol and a model α-hydroxy ester.

A short stereoselective sequence is described for the preparation of the racemic form of the highly functionalized cyclohexene derivative 2 which comprises the upper segment of tetronolide ( 1 ), the aglycone of the antitumor tetrocarcins. A key element of this route is rapid assembly and intramolecular cycloaddition of the trienyl enol pyruvate 4 , which achieves complete control over regiochemistry and good stereochemical control.

A one-pot two-step phosphorylation procedure is described which is suitable for the preparation of thermally lacible or highly substituted dialkyl phosphonates for use in Horner-Emmons olefinations. Such systems are not readily available using the traditional methods such as the Arbuzov reaction or acylation of dialkyl phosphonate carbanions. Reaction of dioxinone lithium enolates with diethyl chlorophosphite followed by oxidation provide the desired phosphonates in good to excellent yields and conversions.

[reaction--see text] [corrected] The preparation and thermolysis of new stable heterocyclic precursors of 1,2-diaza-1,3-butadienes is described. The resulting reactive diazadienes are trapped in situ with N-phenylmaleimide [corrected]. The effect of precursor structure on the temperature at which the diazadienes are generated is discussed.

The ring systems present in the two major classes of ophiobolane sesterterpenes have been obtained via a common bicyclic intermediate. In each case, the eight-membered ring was constructed by fragmentation of an appropriately functionalized bicyclo(3.3.1)nonane ring system.

L'introduction d'un groupe trimethylsilyl a des positions determinees dans des substrats trieniques subissant une cyclisation par cycloaddition Diels Alder intramoleculaire produisant des derives de l'indane ou de l'octahydronaphtalene conduit a une augmentation du niveau de diastereoselectivite