A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Large-Scale Virtual Screening for the Discovery of SARS-CoV-2 Papain-like Protease (PLpro) Non-covalent Inhibitors.

The rapid global spread of the SARS-CoV-2 virus facilitated the development of novel direct-acting antiviral agents (DAAs). The papain-like protease (PLpro) has been proposed as one of the major SARS-CoV-2 targets for DAAs due to its dual role in processing viral proteins and facilitating the host's immune suppression. This dual role makes identifying small molecules that can effectively neutralize SARS-CoV-2 PLpro activity a high-priority task. However, PLpro drug discovery faces a significant challenge due to the high mobility and induced-fit effects in the protease's active site. Herein, we virtually screened the ZINC20 database with Deep Docking (DD) to identify prospective noncovalent PLpro binders and combined ultra-large consensus docking with two pharmacophore (ph4)-filtering strategies. The analysis of active compounds revealed their somewhat-limited diversity, likely attributed to the induced-fit nature of PLpro's active site in the crystal structures, and therefore, the use of rigid docking protocols poses inherited limitations. The top hits were assessed against recombinant viral proteins and live viruses, demonstrating desirable inhibitory activities. The best compound VPC-300195 (IC50: 15 µM) ranks among the top noncovalent PLpro inhibitors discovered through in silico methodologies. In the search for novel SARS-CoV-2 PLpro-specific chemotypes, the identified inhibitors could serve as diverse templates for the development of effective noncovalent PLpro inhibitors.

Lipid nanoparticle-mediated silencing of osteogenic suppressor GNAS leads to osteogenic differentiation of mesenchymal stem cells in vivo.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest. Here we examine the ability of small interfering RNA (siRNA) to enhance osteoblast differentiation and bone formation by silencing the negative suppressor gene GNAS in bone MSCs. Using clinically validated lipid nanoparticle (LNP) siRNA delivery systems, we show that silencing the suppressor gene GNAS in vitro in MSCs leads to molecular and phenotypic changes similar to those seen in osteoblasts. Further, we demonstrate that these LNP-siRNAs can transfect a large proportion of mice MSCs in the compact bone following intravenous injection. Transfection of MSCs in various animal models led to silencing of GNAS and enhanced differentiation of MSCs into osteoblasts. These data demonstrate the potential for LNP delivery of siRNA to enhance the differentiation of MSCs into osteoblasts, and suggests that they are a promising approach for the treatment of osteoporosis and other bone diseases.

A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes-based on the structure of ivacaftor-to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021).

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis.

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.

Design, Synthesis, Pharmacokinetics, and Biodistribution of a Series of Bone-Targeting EP4 Receptor Agonist Prodrugs for Treatment of Osteoporosis and Other Bone Conditions.

A series of bone-targeting EP4 receptor agonist conjugate prodrugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Singly and doubly radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream, and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up biodistribution and elimination studies. Doubly radiolabeled conjugate 4 was found to partition largely to the liver and bones, and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from bone-bound 4 with a half-time of about 7 days. When dosed orally, radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged, and only traces of radiolabeled 4 were found in the liver, blood, or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate, forming a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

Effects of treatment with a bone-targeted prostaglandin E2 receptor 4 agonist C3 (Mes-1007) in a mouse model of severe osteogenesis imperfecta.

OBJECTIVE: Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually caused by mutations affecting collagen type I synthesis in osteoblasts. Bisphosphonates are widely used to decrease fracture rate but are only partially effective. Bone anabolic compounds, such as prostaglandin E2 receptor 4 (EP4) agonists may be an alternative treatment approach. Here we assessed the effect of Mes-1007, a novel bone-targeted EP4 agonist in Jrt mice, a model of severe OI. STUDY DESIGN: Experimental study. RESULTS: Male 8-week old wild type (WT) and OI mice were randomly assigned to 4 weeks of three intraperitoneal injections per week with Mes-1007 (25 mg per kg body mass), phosphate-buffered saline, zoledronate (5 µg per kg), or a combination treatment of zoledronate and Mes-1007. Treatment with Mes-1007 alone did not lead to higher trabecular bone volume per tissue volume (BV/TV) in the distal femur or lumbar vertebra 4 in either WT or OI mice. Treatment with zoledronate alone was associated with a significant increase in distal femur and vertebra BV/TV in both genotypes. In zoledronate-treated WT and OI mice, Mes-1007 increased bone formation rate in vertebral trabecular bone and had an additive effect on BV/TV. Vertebral BV/TV in OI mice that received zoledronate or Mes-1007/zoledronate combination treatment was similar to untreated WT mice (p = 0.25). At the femoral midshaft, Mes-1007/zoledronate combination treatment increased cortical thickness in both genotypes and led to higher periosteal diameter in OI mice. Three-point bending tests of femurs showed that Mes-1007/zoledronate combination treatment increased the stiffness, load at yield and maximal load in WT but not in OI mice. CONCLUSION: Dosing Mes-1007 in combination with zoledronate improved the bone properties in a manner that is consistent with a mechanism of action of EP4 agonists on bone and additive to effects of anti-resorptives typified by zoledronate.

Achieving enhanced bone regeneration using monetite granules with bone anabolic drug conjugates (C3 and C6) in rat mandibular defects.

Bone grafting procedures are commonly used to manage bone defects in the craniofacial region. Monetite is an excellent biomaterial option for bone grafting, however, it is limited by lack of osteoinduction. Several molecules can be incorporated within the monetite matrix to promote bone regeneration. The aim was to investigate whether incorporating bone forming drug conjugates (C3 and C6) within monetite can improve their ability to regenerate bone in bone defects. Bilateral bone defects were created in the mandible of 24 Sprague-Dawley rats and were then packed with monetite control, monetite+C3 or monetite+C6. After 2 and 4 weeks, post-mortem samples were analyzed using microcomputed tomography, histology and back-scattered electron microscopy to calculate the percentages of bone formation and remaining graft material. At 2 and 4 weeks, monetite with C3 and C6 demonstrated higher bone formation than monetite control, while monetite+C6 had the highest bone formation percentage at 4 weeks. There were no significant differences in the remaining graft material between the groups at 2 or 4 weeks. Incorporating these anabolic drug conjugates within the degradable matrix of monetite present a promising bone graft alternative for bone regeneration and repair in orthopedic as well as oral and maxillofacial applications.

Improved bone regeneration using bone anabolic drug conjugates (C3 and C6) with deproteinized bovine bone mineral as a carrier in rat mandibular defects.

BACKGROUND: Deproteinized bovine bone mineral (DBBM) has been extensively studied and used for bone regeneration in oral and maxillofacial surgery. However, it lacks an osteoinductive ability. We developed two novel bone anabolic conjugated drugs, known as C3 and C6, of an inactive bisphosphonate and a bone activating synthetic prostaglandin agonist. The aim was to investigate whether these drugs prebound to DBBM granules have the potential to achieve rapid and enhanced bone regeneration. METHODS: Bilateral defects (4.3 mm diameter circular through and through) were created in mandibular angles of 24 Sprague-Dawley rats were filled with DBBM Control, DBBM with C3 or DBBM with C6 (n = 8 defects per group/ each timepoint). After 2 and 4 weeks, postmortem samples were analyzed by microcomputed tomography followed by backscattering electron microscopy and histology. RESULTS: DBBM grafts containing the C3 and C6 conjugated drugs showed significantly more bone formation than DBBM control at 2 and 4 weeks. The C6 containing DBBM demonstrated the highest percentage of new bone formation at 4 weeks. There was no significant difference in the percentage of the remaining graft between the different groups at 2 or 4 weeks. CONCLUSIONS: DBBM granules containing conjugated drugs C3 and C6 induced greater new bone volume generated and increased the bone formation rate more than the DBBM controls. This is expected to allow the development of clinical treatments that provide more predictable and improved bone regeneration for bone defect repair in oral and maxillofacial surgery.

In Vivo Bone Effects of a Novel Bisphosphonate-EP4a Conjugate Drug (C3) for Reversing Osteoporotic Bone Loss in an Ovariectomized Rat Model.

Pathological bone loss is a regular feature of postmenopausal osteoporosis, and the microstructural changes along with the bone loss make the individual prone to getting hip, spine, and wrist fractures. We have developed a new conjugate drug named C3, which has a synthetic, stable EP4 agonist (EP4a) covalently linked to an inactive alendronate (ALN) that binds to bone and allows physiological remodeling. After losing bone for 12 weeks, seven groups of rats were treated for 8 weeks via tail-vein injection. The groups were: C3 conjugate at low and high doses, vehicle-treated ovariectomy (OVX) and sham, C1 (a similar conjugate, but with active ALN at high dose), inactive ALN alone, and a mixture of unconjugated ALN and EP4a to evaluate the conjugation effects. Bone turnover was determined by dynamic and static histomorphometry; µCT was employed to determine bone microarchitecture; and bone mechanical properties were evaluated via biomechanical testing. Treatment with C3 significantly increased trabecular bone volume and vertebral BMD versus OVX controls. There was also significant improvement in the vertebral load-bearing abilities and stimulation of bone formation in femurs after C3 treatment. This preclinical research revealed that C3 resulted in significant anabolic effects on trabecular bone, and EP4a and ALN conjugation components are vital to conjugate anabolic efficacy. A combined therapy using an EP4 selective agonist anabolic agent linked to an inactive ALN is presented here that produces significant anabolic effects, allows bone remodeling, and has the potential for treating postmenopausal osteoporosis or other diseases where bone strengthening would be beneficial. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

A Novel Anabolic Conjugate (C3) in the Matrix of Dicalcium Phosphate Onlay Block Grafts for Achieving Vertical Bone Augmentation: An Experimental Study on Rabbit Calvaria.

PURPOSE: Achieving successful and predictable alveolar ridge augmentation in the vertical dimension is extremely challenging. Several materials have been investigated to achieve vertical ridge augmentation; however, the results are highly unpredictable. The collaborative team presenting this research has developed brushite- and monetite-based grafts that incorporate in their matrix a novel bone anabolic conjugate (C3) of a bisphosphonate and a potent bone-activating EP4 receptor agonist. The study objective was to investigate the potential of these graft formulations to achieve rapid, enhanced, and clinically significant bone regeneration in the vertical dimension. MATERIALS AND METHODS: Brushite and monetite grafts were fabricated and characterized for phase purity, porosity, compressive strength, and microstructural morphology. They were implanted in 12 rabbit calvaria for 12 weeks. Each group (n = 6): brushite control, brushite with C3, monetite control, and monetite with C3. Postmortem samples were retrieved and processed for analysis. The percentage bone volume, vertical bone height gained, and graft resorption were calculated and assessed. RESULTS: The brushite and monetite grafts containing C3 integrated well onto the calvarial bone surface, with new bone extending through the graft area (36% and 80%, respectively), while the C3 lacking grafts showed decreased surface integration and bone infiltration (28% and 38%, respectively). The C3 containing brushite and monetite grafts demonstrated bone growth vertically (1.8 mm and 2.7 mm, respectively) and improved graft resorption. CONCLUSION: The brushite- and monetite-based grafts loaded with the C3 conjugate resulted in greater de novo bone formation in the vertical dimension when compared with the grafts without the drug. However, the monetite grafts produced much more and predictable vertical height gain than was achieved with brushite grafts. The advantages of this new graft drug formulation in future would be to provide more predictable vertical bone regeneration, which will ultimately benefit patients undergoing dental implant placement.

Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.

Targeting therapeutics to bone by conjugation with bisphosphonates.

Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This targeting ability has been exploited to design and prepare bisphosphonate conjugate prodrugs to deliver a wide variety of drug molecules selectively to bones. It is important that conjugates be stable in the blood stream and that conjugate that is not taken up by bone is eliminated rapidly. The prodrugs should release active drug at a rate appropriate so as to provide efficacy. Radiolabelling is the best method to quantify and evaluate pharmacokinetics, tissue distribution, bone uptake and release of the active drug(s). Recent reports have described bisphosphonate conjugates derived from the antiresorptive drug, alendronic acid and anabolic prostanoid drugs that effectively deliver prostaglandins and prostaglandin EP4 receptor agonists to bone and show enhanced anabolic efficacy and tolerability compared to the drugs alone. These conjugate drugs can be dosed infrequently (weekly or bimonthly) whereas the free drugs must be dosed daily.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Further investigation of inhibitors of MRSA pyruvate kinase: Towards the conception of novel antimicrobial agents.

Several novel series of compounds were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as a highly interconnected essential 'hub' protein in MRSA, with structural features distinct from the human homologs which makes it a novel antimicrobial target. Several MRSA PK inhibitors (including the hydrazide 1) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to human PK isoforms. Structure-activity relationship (SAR) studies were carried out on the replacement of the hydrazide linker with 3-atoms, 2-atoms and 0-atom linkers and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 µg/mL.

Targeting Binding Function-3 of the Androgen Receptor Blocks Its Co-Chaperone Interactions, Nuclear Translocation, and Activation.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3-directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936-45. ©2016 AACR.

Precision autophagy: Will the next wave of selective autophagy markers and specific autophagy inhibitors feed clinical pipelines?

Research presented at the Vancouver Autophagy Symposium (VAS) 2014 suggests that autophagy's influence on health and disease depends on tight regulation and precision targeting of substrates. Discussions recognized a pressing need for robust biomarkers that accurately assess the clinical utility of modulating autophagy in disease contexts. Biomarker discovery could flow from investigations of context-dependent triggers, sensors, and adaptors that tailor the autophagy machinery to achieve target specificity. In his keynote address, Dr. Vojo Deretic (University of New Mexico) described the discovery of a cargo receptor family that utilizes peptide motif-based cargo recognition, a mechanism that may be more precise than generic substrate tagging. The keynote by Dr. Alec Kimmelman (Harvard Medical School) emphasized that unbiased screens for novel selective autophagy factors may accelerate the development of autophagy-based therapies. Using a quantitative proteomics screen for de novo identification of autophagosome substrates in pancreatic cancer, Kimmelman's group discovered a new type of selective autophagy that regulates bioavailable iron. Additional presentations revealed novel autophagy regulators and receptors in metabolic diseases, proteinopathies, and cancer, and outlined the development of specific autophagy inhibitors and treatment regimens that combine autophagy modulation with anticancer therapies. VAS 2014 stimulated interdisciplinary discussions focused on the development of biomarkers, drugs, and preclinical models to facilitate clinical translation of key autophagy discoveries.

Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B.

An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B. Substrate cleavage was readily monitored and was linear for up to 4h but enzyme concentrations of about ten-fold higher were required compared to assays using protein substrate LC3 or analogs thereof (such as FRET-LC3). Several known inhibitors of ATG4B were evaluated using the small peptide substrate and gave IC50 values 3-7 fold higher than previously obtained values using the FRET-LC3 substrate.

Determination of the rat in vivo pharmacokinetic profile of a bone-targeting dual-action pro-drug for treatment of osteoporosis.

The in vivo hydrolytic pathway of a dual-function bone-targeting EP4 receptor agonist-bisphosphonate pro-drug was deduced from radiolabeling experiments. A (14)C labeled pro-drug was used to monitor liberation of the bisphosphonate and results were compared to parallel studies where the EP4 receptor agonist was labeled with (3)H. The bone-adsorption of the (14)C pro-drug following an IV bolus was about 10% compared to 7.8% for the tritiated pro-drug. The difference in release half-life (5.2 and 19.7 days from (3)H and (14)C experiments, respectively) indicated that, after binding to bone, the initial hydrolysis occurred at the ester moiety of the linker releasing the EP4 agonist. The conjugate was found to concentrate in more porous, high-surface-area regions of the long bones. Both (3)H and (14)C experiments indicated a short circulating half-life (1-2 h) in blood.