Subthreshold activation of the melanocortin system causes generalized sensitization to anorectic agents in mice.

The melanocortin-3 receptor (MC3R) regulates GABA release from agouti-related protein (AgRP) nerve terminals and thus tonically suppresses multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of the MC3R and the melanocortin system in regulating the response to various anorexigenic agents. The genetic deletion or pharmacological inhibition of the MC3R, or subthreshold doses of an MC4R agonist, improved the dose responsiveness to glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs).

Inhibition of the melanocortin-3 receptor (MC3R) causes generalized sensitization to anorectic agents.

The melanocortin-3 receptor (MC3R) acts presynaptically to regulate GABA release from agouti-related protein (AgRP) nerve terminals and thus may be a negative regulator of multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of MC3R in regulating the response to various anorexigenic agents. Our findings reveal that genetic deletion or pharmacological inhibition of MC3R improves the dose responsiveness to Glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss.

Inhibition of CREB Binding and Function with a Dual-Targeting Ligand.

CBP/p300 is a master transcriptional coactivator that regulates gene activation by interacting with multiple transcriptional activators. Dysregulation of protein-protein interactions (PPIs) between the CBP/p300 KIX domain and its activators is implicated in a number of cancers, including breast, leukemia, and colorectal cancer. However, KIX is typically considered "undruggable" because of its shallow binding surfaces lacking both significant topology and promiscuous binding profiles.

C-H radiocyanation of bioactive molecules sequential iodination/copper-mediated cross-coupling.

This report describes a net C-H radiocyanation reaction for the transformation of electron rich (hetero)aromatic substrates into CN-labeled products. Electrophilic C(sp)-H iodination of the (hetero)arene with -iodosuccinimide is followed by Cu-mediated radiocyanation with KCN. This sequence is applied to a variety of substrates, including the nucleobases uracil and cytosine, the amino acids tyrosine and tryptophan, and the peptide LYRAGWRAFS, which undergoes selective C-H radiocyanation at the tryptophan (W) residue.

Garcinolic Acid Distinguishes Between GACKIX Domains and Modulates Interaction Networks.

Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (K 1 μM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX.

TMPRSS2 Inhibitor Discovery Facilitated through an and Biochemical Screening Platform.

The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a promising antiviral target because it plays a role in priming the spike protein before viral entry occurs for the most virulent variants. Further, TMPRSS2 has no established physiological role, thereby increasing its attractiveness as a target for antiviral agents.

A lipopeptidomimetic of transcriptional activation domains selectively disrupts Med25 PPIs.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (34913-8) increases the affinity for the coactivator Med25 >10-fold ( >>100 μM to 10 μM).

Allostery in the dynamic coactivator domain KIX occurs through minor conformational micro-states.

The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator•coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb•KIX•MLL and pKID•KIX•MLL.

Copper-Mediated Radiocyanation of Unprotected Amino Acids and Peptides.

This report describes a copper-mediated radiocyanation of aryl halides that is applicable to complex molecules. This transformation tolerates an exceptionally wide range of functional groups, including unprotected amino acids. As such, it enables the site-specific introduction of [C]CN into peptides at an iodophenylalanine residue.

A Dual-Site Inhibitor of CBP/p300 KIX is a Selective and Effective Modulator of Myb.

The protein-protein interaction between the KIX motif of the transcriptional coactivator CBP/p300 and the transcriptional activator Myb is a high-value target due to its established role in certain acute myeloid leukemias (AML) and potential contributions to other cancers. However, the CBP/p300 KIX domain has multiple binding sites, several structural homologues, many binding partners, and substantial conformational plasticity, making it challenging to specifically target using small-molecule inhibitors. Here, we report a picomolar dual-site inhibitor (MybLL-tide) of the Myb-CBP/p300 KIX interaction.

Norstictic Acid Is a Selective Allosteric Transcriptional Regulator.

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery.

The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia.

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans.

TMPRSS2 inhibitor discovery facilitated through an and biochemical screening platform.

The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role.

Unexpected specificity within dynamic transcriptional protein-protein complexes.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model.

Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation of IKKα.

The small molecule gibberellin JRA-003 was identified as an inhibitor of the NF-kB (nuclear kappa-light-chain-enhancer of activated B cells) pathway. Here we find that JRA-003 binds to and significantly inhibits the nuclear translocation of pathway-activating kinases IKKα (IκB kinase alpha) and IKKβ (IκB kinase beta). Analogs of JRA-003 were synthesized and NF-κB-inhibiting gibberellins were found to be cytotoxic in cancer-derived cell lines (HS 578T, HCC 1599, RC-K8, Sud-HL4, CA 46, and NCIH 4466).

Selective Modulation of Dynamic Protein Complexes.

Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs.

Roles for a lipid phosphatase in the activation of its opposing lipid kinase.

Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14.

Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions.

The photoactivatable amino acid p-benzoyl-l-phenylalanine (pBpa) has been used for the covalent capture of protein-protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein-protein interactions.

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain.

The kinase-inducible domain (KIX) of the transcriptional coactivator CBP binds multiple transcriptional regulators through two allosterically connected sites. Establishing a method for observing activator-specific KIX conformations would facilitate the discovery of drug-like molecules that capture specific conformations and further elucidate how distinct activator-KIX complexes produce differential transcriptional effects. However, the transient and low to moderate affinity interactions between activators and KIX are difficult to capture using traditional biophysical assays.

Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators.

Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a β-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences.

Pharmaceutical solvate formation for the incorporation of the antimicrobial agent hydrogen peroxide.

Antimicrobial functionality is introduced into a pharmaceutical formulation of miconazole while improving solubility. The work leverages hydrate formation propensity in order to produce hydrogen peroxide solvates. The ubiquity of hydrate formation suggests that hydrogen peroxide can be broadly deployed in pharmaceuticals, rendering a liquid excipient suitable for solid pharmaceutical formulations.

Modulating the masters: chemical tools to dissect CBP and p300 function.

Dysregulation of transcription is found in nearly every human disease, and as a result there has been intense interest in developing new therapeutics that target regulators of transcription. CREB binding protein (CBP) and its paralogue p300 are attractive targets due to their function as `master coactivators'. Although inhibitors of several CBP/p300 domains have been identified, the selectivity of many of these compounds has remained underexplored.

Covalent Chemical Cochaperones of the p300/CBP GACKIX Domain.

The GACKIX activator binding domain has been a compelling target for small-molecule probe discovery because of the central role of activator-GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context-dependent function of activator-coactivator complexes. However, the dynamic and transient protein-protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules.

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability.

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t = 39.