Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by 19F NMR and Cryo-EM.

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the ß5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum-infected mice.

Protocol for analysis of intracellular conversion of artezomib molecules into new proteasome inhibitors in Plasmodium falciparum parasites.

Artezomibs (ATZs), dual-pharmacophore molecules comprising of artemisinin and a parasite proteasome inhibitor, hijack parasite ubiquitin proteasome system to transform into new proteasome inhibitors following the activation of artemisinin by heme.1 Here, we present a protocol for using a fluorescent activity-based broad-spectrum proteasome inhibitor probe to study intracellular conversion of ATZ molecules into new proteasome inhibitors in malaria parasites. We describe steps for drug treatment and washout, parasite lysis, proteasome labeling, and visualization. For complete details on the use and execution of this protocol, please refer to Zhan et al.1.

Directly Suppressing MYC Function with Novel Alkynyl-Substituted Phenylpyrazole Derivatives that Induce Protein Degradation and Perturb MYC/MAX Interaction.

Despite being a highly sought-after therapeutic target for human malignancies, myelocytomatosis viral oncogene homologue (MYC) has been considered intractable due to its intrinsically disordered nature, making the discovery of in vivo effective inhibitors that directly block its function challenging. Herein, we report structurally novel alkynyl-substituted phenylpyrazole derivatives directly perturbing MYC function. Among them, compound 37 exhibited superior antiproliferative activities to those of MYCi975 against multiple malignant cell lines. It induced dose-dependent MYC degradation in cells with degradation observed at the concentration as low as 1.0 µM. Meanwhile, its direct suppression of MYC function was confirmed by the capability to inhibit the binding of MYC/MYC-associated protein X (MAX) heterodimer to DNA consensus sequence, induce MYC thermal instability, and disturb MYC/MAX interaction. Moreover, 37 demonstrated enhanced therapeutic efficacy over MYCi975 in a mouse allograft model of prostate cancer. Overall, 37 deserves further development for exploring MYC-targeting cancer therapeutics.

Exploitation of Proximity-Mediated Effects in Drug Discovery: An Update of Recent Research Highlights in Perturbing Pathogenic Proteins and Correlated Issues.

The utilization of proximity-mediated effects to perturb pathogenic proteins of interest (POIs) has emerged as a powerful strategic alternative to conventional drug design approaches based on target occupancy. Over the past three years, the burgeoning field of targeted protein degradation (TPD) has witnessed the expansion of degradable POIs to membrane-associated, extracellular, proteasome-resistant, and even microbial proteins. Beyond TPD, researchers have achieved the proximity-mediated targeted protein stabilization, the recruitment of intracellular immunophilins to disturb undruggable targets, and the nonphysiological post-translational modifications of POIs. All of these strides provide new avenues for innovative drug discovery aimed at battling human malignancies and other major diseases. This perspective presents recent research highlights and discusses correlated issues in developing therapeutic modalities that exploit proximity-mediated effects to modulate pathogenic proteins, thereby guiding future academic and industrial efforts in this field.

Discovery of Highly Selective Inhibitors of the Human Constitutive Proteasome ß5c Chymotryptic Subunit.

We describe our discovery and development of potent and highly selective inhibitors of human constitutive proteasome chymotryptic activity (ß5c). Structure-activity relationship studies of the novel class of inhibitors focused on optimization of N-cap, C-cap, and side chain of the chemophore asparagine. Compound 32 is the most potent and selective ß5c inhibitor in this study. A docking study provides a structure rationale for potency and selectivity. Kinetic studies show a reversible and noncompetitive inhibition mechanism. It enters the cells to engage the proteasome target, potently and selectively kills multiple myeloma cells, and does so by synergizing with a ß5i-selective inhibitor.

Structure of the human UBR5 E3 ubiquitin ligase.

The human UBR5 is a single polypeptide chain homology to E6AP C terminus (HECT)-type E3 ubiquitin ligase essential for embryonic development in mammals. Dysregulated UBR5 functions like an oncoprotein to promote cancer growth and metastasis. Here, we report that UBR5 assembles into a dimer and a tetramer. Our cryoelectron microscopy (cryo-EM) structures reveal that two crescent-shaped UBR5 monomers assemble head to tail to form the dimer, and two dimers bind face to face to form the cage-like tetramer with all four catalytic HECT domains facing the central cavity. Importantly, the N-terminal region of one subunit and the HECT of the other form an "intermolecular jaw" in the dimer. We show the jaw-lining residues are important for function, suggesting that the intermolecular jaw functions to recruit ubiquitin-loaded E2 to UBR5. Further work is needed to understand how oligomerization regulates UBR5 ligase activity. This work provides a framework for structure-based anticancer drug development and contributes to a growing appreciation of E3 ligase diversity.

Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors.

The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S ß6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone ß2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sß6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds ß2 and ß5 in wild type Pf20S as well as WLW-vs binds ß2 and ß5 in Pf20Sß6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance.

Structure-Activity Relationship Studies of Antimalarial Plasmodium Proteasome Inhibitors─Part II.

With increasing reports of resistance to artemisinins and artemisinin-combination therapies, targeting the Plasmodium proteasome is a promising strategy for antimalarial development. We recently reported a highly selective Plasmodium falciparum proteasome inhibitor with anti-malarial activity in the humanized mouse model. To balance the permeability of the series of macrocycles with other drug-like properties, we conducted further structure-activity relationship studies on a biphenyl ether-tethered macrocyclic scaffold. Extensive SAR studies around the P1, P3, and P5 groups and peptide backbone identified compound TDI-8414. TDI-8414 showed nanomolar antiparasitic activity, no toxicity to HepG2 cells, high selectivity against the Plasmodium proteasome over the human constitutive proteasome and immunoproteasome, improved solubility and PAMPA permeability, and enhanced metabolic stability in microsomes and plasma of both humans and mice.

Dual-pharmacophore artezomibs hijack the Plasmodium ubiquitin-proteasome system to kill malaria parasites while overcoming drug resistance.

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome.

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

Design, synthesis and biological evaluation of novel acrylamide analogues as inhibitors of BCR-ABL kinase.

A series of acrylamide analogues were designed and synthesized from Imatinib and Nilotinib as novel BCR-ABL inhibitors by application of the principle of nonclassical electronic isostere. All new compounds were evaluated for their inhibitory effects on the activity of BCR-ABL kinase and the proliferation of K562 leukemia cancer cells in vitro. The acrylamide analogues in which the substituent in C ring was trifluoromethyl group were identified as highly potent BCR-ABL kinase inhibitors. Compound 13f exhibited an IC(50) value as low as 20.6 nM in ABL kinase inhibition and an IC(50) value of 32.3 nM for antiproliferative activity, about 10.5-fold and 12-fold lower than those of Imatinib respectively. These results suggest that compound 13f is a promising candidate as a novel BCR-ABL kinase inhibitor for further development.

Design, synthesis and antitumor activities of novel bis-aryl ureas derivatives as Raf kinase inhibitors.

A series of novel bis-aryl ureas containing trifluoromethyl imidazolyl group targeting Raf kinase were designed and synthesized based on the lead compound of Sorafenib. All the prepared compounds were evaluated for their in vitro antiproliferative activities against three human cancer cell lines including MDA-MB-231 (breast), BGC-823 (gastric), and SMMC-7721 (liver). Several compounds from the series exhibited excellent antitumor activities against all three tested cancer lines. Further their inhibitory activities against Raf kinase were investigated, and three compounds (11c, 11d, and 11p) demonstrated better activities than contrast drug Sorafenib. Especially compound 11c was found to be a potent and selective Raf kinase inhibitor and could be considered as a candidate compound for further development.

Design, Synthesis, and Optimization of Macrocyclic Peptides as Species-Selective Antimalaria Proteasome Inhibitors.

With over 200 million cases and close to half a million deaths each year, malaria is a threat to global health, particularly in developing countries. Plasmodium falciparum, the parasite that causes the most severe form of the disease, has developed resistance to all antimalarial drugs. Resistance to the first-line antimalarial artemisinin and to artemisinin combination therapies is widespread in Southeast Asia and is emerging in sub-Saharan Africa. The P. falciparum proteasome is an attractive antimalarial target because its inhibition kills the parasite at multiple stages of its life cycle and restores artemisinin sensitivity in parasites that have become resistant through mutation in Kelch K13. Here, we detail our efforts to develop noncovalent, macrocyclic peptide malaria proteasome inhibitors, guided by structural analysis and pharmacokinetic properties, leading to a potent, species-selective, metabolically stable inhibitor.

Discovery of N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide (Hu7691), a Potent and Selective Akt Inhibitor That Enables Decrease of Cutaneous Toxicity.

Rash is one of the primary dose-limiting toxicities of Akt (protein kinase B) inhibitors in clinical trials. Here, we demonstrate the inhibition of Akt2 isozyme may be a driver for keratinocyte apoptosis, which promotes us to search for new selective Akt inhibitors with an improved cutaneous safety property. According to our previous research, compound 2 is selected for further optimization for overcoming the disadvantages of compound 1, including high Akt2 inhibition and high toxicity against HaCaT keratinocytes. The dihedral angle-based design and molecular dynamics simulation lead to the identification of Hu7691 (B5) that achieves a 24-fold selectivity between Akt1 and Akt2. Hu7691 exhibits low activity in inducing HaCaT apoptosis, promising kinase selectivity, and excellent anticancer cell proliferation potencies. Based on the superior results of safety property, pharmacokinetic profile, and in vivo efficacy, the National Medical Products Administration (NMPA) approved the investigational new drug (IND) application of Hu7691.

Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome.

Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6. The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.

Structure-Activity Relationships of Noncovalent Immunoproteasome ß5i-Selective Dipeptides.

The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic ß5i subunit of i-20S inhibits T cell activation, B cell proliferation, and dendritic cell differentiation in vitro and suppresses immune responses in animal models of autoimmune disorders and allograft rejection. However, cytotoxicity to immune cells has accompanied the use of covalently reactive ß5i inhibitors, whose activity against the constitutive proteasome (c-20S) is cumulative with the time of exposure. Herein, we report a structure-activity relationship study of a class of noncovalent proteasome inhibitors with picomolar potencies and 1000-fold selectivity for i-20S over c-20S. Furthermore, these inhibitors are specific for ß5i over the other five active subunits of i-20S and c-20S, providing useful tools to study the functions of ß5i in immune responses. The potency of these compounds in inhibiting human T cell activation suggests that they may have therapeutic potential.

Discovery of pyrazole-thiophene derivatives as highly Potent, orally active Akt inhibitors.

A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound 1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft model. Compound 1o might be a potential candidate for further development.

Phenotypic Screening-Based Identification of 3,4-Disubstituted Piperidine Derivatives as Macrophage M2 Polarization Modulators: An Opportunity for Treating Multiple Sclerosis.

Multiple sclerosis (MS) is a disease of the autoimmune-mediated disorder in the central nervous system, for which no effective therapeutic agent is currently available. The regulation of macrophage polarization toward M2 is a general benefit for treating MS. The gene biomarker-based phenotypic screening approach was developed, and 3,4-disubstituted piperidine derivative S-28 was identified as a lead compound modulating macrophage M2 polarization. Further SAR studies resulted in the discovery of the most potent modulator D11 that showed good oral bioavailability and significant in vivo therapeutic effects. Mechanistic studies demonstrated that the M2 polarization macrophages modulated by D11 mainly functioned through inhibiting the proliferation of T-cells and activating the phosphorylation of Stat3 and Akt. Therefore, the gene biomarker-based phenotypic screening was demonstrated as a promising tool for the discovery of novel macrophage M2 polarization modulators. Compound D11 may serve as a promising starting point for the development of therapeutics to treat MS.