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Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5′-monophosphate–mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid–sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5′-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum , namely tyrosine RS ( Pf YRS). ML901 exerts whole-life-cycle–killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

Most EGFR exon 20 insertion (EGFRex20ins) driver mutations in non–small cell lung cancer (NSCLC) are insensitive to approved EGFR tyrosine kinase inhibitors (TKI). To address the limitations of existing therapies targeting EGFR-mutated NSCLC, mobocertinib (TAK-788), a novel irreversible EGFR TKI, was specifically designed to potently inhibit oncogenic variants containing activating EGFRex20ins mutations with selectivity over wild-type EGFR. The in vitro and in vivo activity of mobocertinib was evaluated in engineered and patient-derived models harboring diverse EGFRex20ins mutations. Mobocertinib inhibited viability of various EGFRex20ins-driven cell lines more potently than approved EGFR TKIs and demonstrated in vivo antitumor efficacy in patient-derived xenografts and murine orthotopic models. These findings support the ongoing clinical development of mobocertinib for the treatment of EGFRex20ins-mutated NSCLC.Significance:No oral EGFR-targeted therapies are approved for EGFR exon 20 insertion (EGFRex20ins) mutation-driven NSCLC. Mobocertinib is a novel small-molecule EGFR inhibitor specifically designed to target EGFRex20ins mutants. Preclinical data reported here support the clinical development of mobocertinib in patients with NSCLC with EGFR exon 20 insertion mutations.See related commentary by Pacheco, p. 1617.This article is highlighted in the In This Issue feature, p. 1601

Supplementary Tables (S1-6) and Figures (S1-6)

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC(50) values

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Significance Here, we describe inhibitors of the Plasmodium proteasome, an enzymatic complex that malaria parasites rely on to degrade proteins. Starting from inhibitors developed to treat cancer, derivatives were designed and synthesized with the aim of increasing potency against the Plasmodium proteasome and decreasing activity against the human enzyme. Biochemical and cellular assays identified compounds that exhibit selectivity and potency, both in vitro and in vivo, at different stages of the parasite’s lifecycle. Cryo-electron microscopy revealed that the inhibitors bind in a hydrophobic pocket that is structurally different in the human proteasome—underpinning their selectivity. The work will help develop antimalarial therapeutics, which are desperately needed to treat a disease that kills nearly half a million people annually., The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax. They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) beta5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

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Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

Introduction: The proteasome is a multi-subunit enzyme complex responsible for the turnover of short-lived, abnormal or damaged proteins in eukaryotic cells. As organisms that undergo rapid growth and cell division, protozoan parasites exist on the knife-edge of proteotoxic catastrophe and thus rely heavily on their protein quality control machinery for survival. Because of this, the proteasome has recently emerged as a desirable drug target.Area covered: This review focuses on efforts to identify protozoan parasite-specific proteasome inhibitors using substrate profiling, library screening, and in vitro evolution of resistance approaches to inform medicinal chemistry. Targeting the parasite's 20S proteasome chymotrypsin-like (β5) activity and selectively inhibiting protein turnover in parasites compared to human cells are critical properties of potent, selective inhibitors.Expert opinion: Proteasome inhibitors have the potential for rapid action against all stages, all species and all strains of plasmodium and kinetoplastid parasites. Given the high level of conservation of proteasome active sites in eukaryotes, an important challenge is achieving inhibitors that show sufficient selectivity while maintaining properties consistent with drug development.

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome β5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.

Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.

Acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide are potent sub-type selective HDAC6 inhibitors. Constrained heterocyclic analogs based on 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine show further enhanced HDAC6 selectivity and inhibitory activity in cells. Homology models suggest that the heterocyclic spacer can more effectively access the wider catalytic channel of HDAC6 compared to other HDAC sub-types.

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.

Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.

Imidazoline‐directed lithiation of 2‐o‐tolyl‐4,5‐dihydro‐1H‐imidazole and its subsequent treatment with electrophiles yields C‐alkylated products, without the need for a nitrogen protecting group. A variety of electrophiles were used to install aromatic, heteroaromatic, and alkyl substituted moieties.

A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae . Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

Purpose: Systematic quality-of-life (QOL) assessment may have value in oncology practice by increasing awareness of a wide range of issues, possibly increasing detection of psychologic morbidity, social problems, and changes in physical status, and improving care and its outcomes. However, logistic problems are substantial. Automated systems solve many of these problems. We field-tested the feasibility and compliance that can be achieved using a computer touchscreen system in two consecutive studies. Patients and Methods: In study 1, a prospective cohort of 272 patients was offered QOL assessment at each clinic appointment for 6 months. In study 2, all patients (N = 1,291) were offered QOL assessment as part of clinic routine during a 12-week period. Results: In study 1, 82% of patients agreed to take part, but over time, compliance was poor (median, 40%; mean, 43%) and deteriorated with longer follow-up. In study 2, the overall compliance was greatly increased (median, 100%; mean, 70%), and compliance was retained over multiple visits. In study 1, compliance was better in younger patients, males, and socially advantaged patients, but was not affected by the presence of depression or anxiety, or QOL. In the second study, building on experience in the first study, data collection and storage in the computer system was excellent, achieving 98% of collected data stored in one center. In general, patients were comfortable with the computers and the approach. Data collection on the wards was more difficult and less complete than in clinics, especially for patients undergoing acute admissions. Conclusion: Feasibility with higher compliance was demonstrated in study 2, in which the data collection was integrated into routine care, and can be improved with further technical initiatives and education of staff.

A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.

The complete relative and absolute stereochemistries have been elucidated for the ansamycin antibiotics (+)-trienomycins A, B, and C and their potent antifungal congeners, the (+)-mycotrienins I and II. A new species, (+)-trienomycin F, has also been isolated and characterized. In addition, an end-game synthetic strategy for the trienomycins and mycotrienins has been developed.

The hydrolytic kinetic resolution (HKR) of terminal epoxides catalyzed by chiral (salen)CoIII complex 1·OAc affords both recovered unreacted epoxide and 1,2-diol product in highly enantioenriched form. As such, the HKR provides general access to useful, highly enantioenriched chiral building blocks that are otherwise difficult to access, from inexpensive racemic materials. The reaction has several appealing features from a practical standpoint, including the use of H2O as a reactant and low loadings (0.2−2.0 mol %) of a recyclable, commercially available catalyst. In addition, the HKR displays extraordinary scope, as a wide assortment of sterically and electronically varied epoxides can be resolved to ≥99% ee. The corresponding 1,2-diols were produced in good-to-high enantiomeric excess using 0.45 equiv of H2O. Useful and general protocols are provided for the isolation of highly enantioenriched epoxides and diols, as well as for catalyst recovery and recycling. Selectivity factors (krel) were determined ...

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.

Imidazoline‐directed lithiation of 2‐o‐tolyl‐4,5‐dihydro‐1H‐imidazole and its subsequent treatment with electrophiles yields C‐alkylated products, without the need for a nitrogen protecting group. A variety of electrophiles were used to install aromatic, heteroaromatic, and alkyl substituted moieties.

The hydrolytic kinetic resolution (HKR) of terminal epoxides catalyzed by chiral (salen)Co(III) complex 1 x OAc affords both recovered unreacted epoxide and 1,2-diol product in highly enantioenriched form. As such, the HKR provides general access to useful, highly enantioenriched chiral building blocks that are otherwise difficult to access, from inexpensive racemic materials. The reaction has several appealing features from a practical standpoint, including the use of H(2)O as a reactant and low loadings (0.2-2.0 mol %) of a recyclable, commercially available catalyst. In addition, the HKR displays extraordinary scope, as a wide assortment of sterically and electronically varied epoxides can be resolved toor = 99% ee. The corresponding 1,2-diols were produced in good-to-high enantiomeric excess using 0.45 equiv of H(2)O. Useful and general protocols are provided for the isolation of highly enantioenriched epoxides and diols, as well as for catalyst recovery and recycling. Selectivity factors (k(rel)) were determined for the HKR reactions by measuring the product ee at ca. 20% conversion. In nearly all cases, k(rel) values for the HKR exceed 50, and in several cases are well in excess of 200.

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a recently identified zinc metalloprotease with carboxypeptidase activity that was identified using our genomics platform. We implemented a rational design approach to identify potent and selective ACE2 inhibitors. To this end, picomolar inhibitors of ACE2 were designed and synthesized.

The intramolecular [4+2] cycloaddition of conjugated enynes provides an efficient and general route to aromatic and dihydroaromatic compounds