Determination of Antibody-Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation.

Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.

Synthesis, molecular editing, and biological assessment of the potent cytotoxin leiodermatolide.

It was by way of total synthesis that the issues concerning the stereostructure of leiodermatolide (1) have recently been solved; with the target now being unambiguously defined, the mission of synthesis changes as to secure a meaningful supply of this exceedingly scarce natural product derived from a deep-sea sponge. To this end, a scalable route of 19 steps (longest linear sequence) has been developed, which features a catalytic asymmetric propargylation of a highly enolizable ß-keto-lactone, a ring closing alkyne metathesis and a modified Stille coupling as the key transformations. Deliberate digression from this robust blueprint brought a first set of analogues into reach, which allowed the lead qualities of 1 to be assessed. The acquired biodata show that 1 is a potent cytotoxin in human tumor cell proliferation assays, distinguished by GI50 values in the ≤3 nM range even for cell lines expressing the Pgp efflux transporter. Studies with human U2OS cells revealed that 1 causes mitotic arrest, micronucleus induction, centrosome amplification and tubulin disruption, even though no evidence for direct tubulin binding has been found in cell-free assays; moreover, the compound does not seem to act through kinase inhibition. Indirect evidence points at centrosome declustering as a possible mechanism of action, which provides a potentially rewarding outlook in that centrosome declustering agents hold promise of being inherently selective for malignant over healthy human tissue.

Borrelidin B: isolation, biological activity, and implications for nitrile biosynthesis.

Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.

Total syntheses and biological reassessment of lactimidomycin, isomigrastatin and congener glutarimide antibiotics.

Lactimidomycin (1) was described in the literature as an exquisitely potent cell migration inhibitor. Encouraged by this claim, we developed a concise and scalable synthesis of this bipartite glutarimide-macrolide antibiotic, which relies on the power of ring-closing alkyne metathesis (RCAM) for the formation of the unusually strained 12-membered head group. Subsequent deliberate digression from the successful path to 1 also brought the sister compound isomigrastatin (2) as well as a series of non-natural analogues of these macrolides into reach. A careful biological re-evaluation of this compound collection showed 1 and progeny to be potently cytotoxic against a panel of cancer cell lines, even after one day of compound exposure; therefore any potentially specific effects on tumor cell migration were indistinguishable from the acute effect of cell death. No significant cell migration inhibition was observed at sub-toxic doses. Although these findings cannot be reconciled with some reports in the literature, they are in accord with the notion that lactimidomycin is primarily a ribosome-binder able to effectively halt protein biosynthesis at the translation stage.

New cytotoxic benzosuberene analogs. Synthesis, molecular modeling and biological evaluation.

In this Letter we describe the synthesis and biological evaluation of new benzosuberene analogs with structural modifications on the B-ring. The focus was initially to probe the chemical space around the B-ring C-8 position. This position was readily available for derivatization chemistry using our recently developed new synthesis for this compound class. Furthermore, we describe two new B-ring analogs, one containing a diene and the other a cyclic ether group. Both new analogs show excellent potencies in tumor cell proliferation assays. In addition, we describe molecular modeling studies that provide a binding rationale for reference compound 8 in the colchicine binding site using the known colchicine crystal structure. We also examine whether the cell based potency data obtained with selected new analogs are supported by modeling results.

Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade.

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

Different roles of liver X receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype.

Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.

Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments.

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952-63. ©2015 AACR.

A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a).

The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.

A novel peroxisome proliferator-activated receptor alpha/gamma dual agonist demonstrates favorable effects on lipid homeostasis.

Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications.

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.

Fluorogenic substrates for high-throughput measurements of endothelial lipase activity.

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.

Discovery and development of dimeric podocarpic acid leads as potent agonists of liver X receptor with HDL cholesterol raising activity in mice and hamsters.

Liver X receptors are nuclear receptors that regulate metabolism of cholesterol. They are activated by oxysterols resulting in increased transcription of the ABCA1 gene, promoting cholesterol efflux and HDL formation. We have identified podocarpic acid anhydride as a 1nM agonist of LXRalpha and beta receptors. Functionally this agonist was over 8-10-fold better activator of LXR receptors compared to one of the natural ligands, 22-(R)-hydroxy cholesterol, in HEK-293 cells. An imide analog increased the level of HDL by 26%, decreased LDL by 10.6%, and increased triglyceride by 51% in hamsters. Discovery, synthesis, SAR and details of the activities of dimers have been described.

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.

A potent synthetic LXR agonist is more effective than cholesterol loading at inducing ABCA1 mRNA and stimulating cholesterol efflux.

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known as ABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXRalpha (NR1H3) and LXRbeta (NR1H2). In transient transactivation assays, APD was approximately 1000-fold more potent, and yielded approximately 6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1 mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatography-mass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.