Beyond Rule-of-five: Permeability Assessment of Semipeptidic Macrocycles.

Compounds beyond the rule-of-five are generating interest as they expand the molecular toolbox for modulating targets previously considered "undruggable". Macrocyclic peptides are an efficient class of molecules for modulating protein-protein interactions. However, predicting their permeability is difficult as they differ from small molecules. Although constrained by macrocyclization, they generally retain some conformational flexibility associated with an enhanced ability to cross biological membranes. In this study, we investigated the relationship between the structure of semi-peptidic macrocycles and their membrane permeability through structural modifications. Based on a scaffold of four amino acids and a linker, we synthesized 56 macrocycles incorporating modifications in either stereochemistry, N-methylation, or lipophilicity and assessed their passive permeability using the parallel artificial membrane permeability assay (PAMPA). Our results show that some semi-peptidic macrocycles have adequate passive permeability even with properties outside the Lipinski rule of five. We found that N-methylation in position 2 and the addition of lipophilic groups to the side chain of tyrosine led to an improvement in permeability with a decrease in tPSA and 3D-PSA. This enhancement could be attributed to the shielding effect of the lipophilic group on some regions of the macrocycle, which in turn, facilitates a favorable macrocycle conformation for permeability, suggesting some degree of chameleonic behavior.

Zinc Fingers 10 and 11 of Miz-1 undergo conformational exchange to achieve specific DNA binding.

Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.

In-depth NMR characterization of Rab4a structure, nucleotide exchange and hydrolysis kinetics reveals an atypical GTPase profile.

Rab4a is a small GTPase associated with endocytic compartments and a key regulator of early endosomes recycling. Gathering evidence indicates that its expression and activation are required for the development of metastases. Rab4a-intrinsic GTPase properties that control its activity, i.e. nucleotide exchange and hydrolysis rates, have not yet been thoroughly studied. The determination of these properties is of the utmost importance to understand its functions and contributions to tumorigenesis. Here, we used the constitutively active (Rab4aQ67L) and dominant negative (Rab4aS22N) mutants to characterize the thermodynamical and structural determinants of the interaction between Rab4a and GTP (GTPγS) as well as GDP. We report the first 1H, 13C, 15N backbone NMR assignments of a Rab GTPase family member with Rab4a in complex with GDP and GTPγS. We also provide a qualitative description of the extent of structural and dynamical changes caused by the Q67L and S22N mutations. Using a real-time NMR approach and the two aforementioned mutants as controls, we evaluated Rab4a intrinsic nucleotide exchange and hydrolysis rates. Compared to most small GTPases such as Ras, a rapid GTP exchange rate along with slow hydrolysis rate were observed. This suggests that, in a cellular context, Rab4a can self-activate and persist in an activated state in absence of regulatory mechanisms. This peculiar profile is uncommon among the Ras superfamily members, making Rab4a an atypical fast-cycling GTPase and may explain, at least in part, how it contributes to metastases.

Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy.

Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.

Determination of Ligand Binding Affinity and Specificity of Purified START Domains by Thermal Shift Assays Using Circular Dichroism.

The use of direct calorimetric methods such as isothermal titration calorimetry for measuring the affinity and specificity of protein-ligand interactions requires large amounts of proteins and ligands. When material is scarce and/or in the absence of calorimeters, thermal Shift Assays (TSA) using Circular Dichroism (CD) or other spectroscopic methods offers an alternative and quantitative method for the determination of apparent or indirect thermodynamical parameters describing the affinity of ligands for proteins. Indeed, the binding constants of ligands (Kb) and other parameters such as the enthalpy and Gibbs free energy of binding may be estimated from the changes in the stability curves ΔGu(T) of a protein in the presence of a ligand. Here we describe the application of two different procedures proposed by Layton and Hellinga et al. (Biochemistry 49:10831-10841, 2010) to evaluate the apparent Kb of testosterone to the START (StAR-related lipid transfer domain) domains.

Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133.

The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.

Gαs protein binds ubiquitin to regulate epidermal growth factor receptor endosomal sorting.

The Gαs subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gαs in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gαs in this sorting step is still poorly characterized. Here, we report that Gαs interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gαs contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in Gαs confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism.

START domain proteins are conserved α/ß helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through (15)N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.

The binding site specificity of STARD4 subfamily: Breaking the cholesterol paradigm.

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain proteins display diverse expression patterns and cellular localisations. They bind a large variety of lipids and sterols and are involved in lipid metabolism, lipid transfer and cell signalling. The START domain tertiary structure is an α-helix/ß-grip fold module of approximately 210 amino acids delimiting an internal cavity forming the binding site. However, the determinants that dictate ligand specificity and the mechanism of ligand entry and exit are ill-defined. Herein, we review and discuss the current knowledge on ligand specificity and binding mechanism of START domains. More specifically, we highlight that the conserved residues of STARD1, STARD3, STARD4, STARD5 and STARD6 START domains binding sterol play an important structural role for the global protein fold, whereas the residues forming the cavity that fits the shape of their respective ligand are divergent, suggesting their participation in ligand specificity. We also explore the potential binding of steroids to STARD6 in the context of ligand selectivity.

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5.

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

STARD5 specific ligand binding: comparison with STARD1 and STARD4 subfamilies.

We present herein a review of our recent results on the characterization of the binding sites of STARD1, STARD5 and STARD6 using NMR and other biophysical techniques. Whereas STARD1 and STARD6 bind cholesterol, no cholesterol binding could be detected for STARD5. However, titration of STARD5 with cholic acid and chenodeoxycholic acid led to specific binding. Using perturbation of the (1)H-(15)N-HSQC spectra and the sequence specific NMR assignments, we identified the amino acids in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Interestingly, these residues are not conserved in STARD1 and STARD6 and could therefore be key structural determinants of the specificity of START domains toward their ligands. We highlight three tissues expressing STARD5 that are affected by bile acids.

(1)H, (13)C, and (15)N backbone chemical shift assignments of StAR-related lipid transfer domain protein 5 (STARD5).

Steroidogenic acute regulatory (StAR)-related lipid transfer proteins possess a START (steroidogenic acute regulatory-related lipid transfer) domain. START domains are conserved protein modules involved in the non-vesicular intracellular transport of lipids and cholesterol in mammals. Fifteen mammalian proteins, divided in five subfamilies, are reported to possess a START domain. Members of the STARD4 subfamily, i.e. STARD4, 5 and 6 are essentially single START domains and are thought to be involved in the intracellular transport of cholesterol. No structure of a cholesterol-bound START domain from this family has been resolved yet. The determination of the structure of such a complex would contribute to a better understanding of the mechanism of ligand binding and transport by START domains, two unresolved aspects of their structural biology. In this context, we have undertaken the structure determination of a ligand-bound form of STARD5 by NMR. Here, we report the (1)H, (13)C and (15)N backbone resonance assignments of the ligand-free STARD5.

StAR-related lipid transfer domain protein 5 binds primary bile acids.

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4-6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [(1)H-(15)N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the (1)H-(15)N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10-(4) M(-1) and 6.3·10(4) M(-1) were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.