Clearance of plasma PCSK9 via the asialoglycoprotein receptor mediated by heterobifunctional ligands.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting the degradation of hepatic LDL receptors (LDLRs). Current therapeutic approaches use antibodies that disrupt PCSK9 binding to LDLR to reduce circulating LDL-C concentrations or siRNA that reduces PCSK9 synthesis and thereby levels in circulation. Recent reports describe small molecules that, like therapeutic antibodies, interfere with PCSK9 binding to LDLR. We report an alternative approach to decrease circulating PCSK9 levels by accelerating PCSK9 clearance and degradation using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, demonstrate binding in vitro and accelerated PCSK9 clearance in vivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

Preclinical discovery and initial clinical data of WVT078, a BCMA × CD3 bispecific antibody.

B-cell maturation antigen (BCMA) is an ideal target in multiple myeloma (MM) due to highly specific expression in malignant plasma cells. BCMA-directed therapies including antibody drug conjugates, chimeric antigen receptor-T cells and bispecific antibodies (BsAbs) have shown high response rates in MM. WVT078 is an anti-BCMA× anti-CD3 BsAb that binds to BCMA with subnanomolar-affinity. It was selected based on potent T cell activation and anti-MM activity in preclinical models with favorable tolerability in cynomolgus monkey. In the ongoing first-in-human phase I dose-escalation study (NCT04123418), 33 patients received intravenous WVT078 once weekly at escalated dosing. At the active doses of 48-250 µg/kg tested to date (n = 26), the overall response rate (ORR) was 38.5% (90% CI: 22.6-56.4%) and the complete response rate (CRR, stringent complete response + complete response) was 11.5%, (90% CI: 3.2-27.2%). At the highest dose level tested, the ORR was 75% (3 of 4 patients). 26 (78.8%) patients reported at least one Grade ≥3 AE and 16 of these AEs were suspected to be drug related. 20 patients (60.6%) experienced cytokine release syndrome. WVT078 has an acceptable safety profile and shows preliminary evidence of clinical activity at doses tested to date.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

Threading a peptide through a peptide: protein loops, rotaxanes, and knots.

Proteins adopt complex folds in nature that typically avoid conformations that are knotted or "threaded" through closed loops. Is this the result of fundamental barriers to folding, or have proteins simply evolved to avoid threaded conformations? Organic synthesis has been used in supramolecular chemistry to install topological links in small molecules. By following these principles, we now show that it is possible to assemble a topologically linked protein complex by threading a linear protein through a cyclic protein to form a [2]pseudo-rotaxane. Subsequent ring closure using native chemical ligation cyclizes the linear protein, forming a [2]heterocatenane. Although the kinetics of protein threading are slower than the folding kinetics of the native protein, threading appears to be a highly efficient process.

MOR209/ES414, a Novel Bispecific Antibody Targeting PSMA for the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ε to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155-65. ©2016 AACR.

Thermodynamics of a designed protein catenane.

Topological linking of proteins is a new approach for stabilizing and controlling the oligomerization state of proteins that fold in an interwined manner. The recent design of a backbone cyclized protein catenane based on the p53tet domain suggested that topological cross-linking provided increased stability against thermal and chemical denaturation. However, the tetrameric structure complicated detailed biophysical analysis of this protein. Here, we describe the design, synthesis and thermodynamic characterization of a protein catenane based on a dimeric mutant of the p53tet domain (M340E/L344K). The formation of the catenane proceeded efficiently, and the overall structure and oligomerization of the domain was not affected by the formation of the topological link. Unfolding and refolding of the catenane was consistent with a two-state process. The topological link stabilized the dimer against thermal and chemical denaturation considerably, raising the apparent melting temperature by 59 degrees C and the midpoint of denaturation by 4.5M GuHCl at a concentration of 50 microM. The formation of the topological link increased the resistance of the dimer to proteolysis. However, the m value decreased by 1.7kcalmol(-1)M(-1), suggesting a decrease in accessible surface area in the unfolded state. This implies that the stabilization from the topological link is largely due to a destabilization of the unfolded state, similar to other cross-links in proteins. Topological linking therefore provides a powerful and orthogonal tool for the stabilization of peptide and protein oligomers.

Internal motions prime cIAP1 for rapid activation.

Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.

Small-molecule ligands of GD2 ganglioside, designed from NMR studies, exhibit induced-fit binding and bioactivity.

Ganglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.

Probing backbone hydrogen bonds in the hydrophobic core of GCN4.

Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.