Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Development and Adjustment of an Algorithm for Identifying Drug-Related Hospital Admissions in Pediatrics.

OBJECTIVE: Adverse drug events (ADEs) in the outpatient pediatric pharmacotherapy can be serious and lead to inpatient admissions. Recent research only focused on ADE identification during hospitalization. The aim of the present study was to develop an algorithm to identify drug-related hospital admissions in pediatrics. METHODS: A systematic literature research was performed, and a pediatric trigger tool for identifying drug-related inpatient admissions was built. The initial version was tested in a sample of 292 patients admitted to a German university children's hospital. Subsequently, the tool was further improved by combining different modules as a novel approach. RESULTS: The obtained algorithm with 39 triggers in 5 modules identified drug-related inpatient admissions at a sensitivity of 95.5% (95% confidence interval [CI], 89.3%-100%) and a specificity of 16.5% (95% CI, 11.9%-21.2%), respectively. After modifications including trigger activation requiring a combination of different modules, specificity increased to 56.9% (95% CI, 50.7%-63.0%). Identifying 36 of 44 ADEs leading to admission, sensitivity remained high (81.8% [95% CI, 70.4%-93.2%]). The overall positive predictive value was 25.2% (95% CI, 18.1%-32.3%). CONCLUSIONS: The algorithm is the first trigger tool to identify ambulant acquired ADEs leading to hospital admission in pediatrics. However, the underlying patient sample is small.Using a larger population for refinement will allow further specifications and reduction in the total amount of triggers and thus signals.

Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.

Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.

Drug Utilisation and Off-Label Use on a German Neonatal Intensive Care Unit: A Retrospective Cohort Study and 10-Year Comparison.

Pharmacotherapy of neonates is complex and marked to a large extent of off-label use. The implementation of the Paediatric Regulation (2007) gave hope for a change in the safety and efficacy for drugs used in neonatal intensive care units (NICU). This study investigates drug utilisation patterns and off-label use in a German neonatal intensive care unit (NICU) in 2014. A 12-months retrospective, observational cohort study was performed at the NICU of the University Children's Hospital Erlangen, Germany. Licensing status was determined using the Summary of Product Characteristics (SmPC). Results are compared with a similar study conducted 10 years earlier. The study included 204 patients (57.8% male) (2004: 183) and 2274 drug prescriptions were recorded (2004: 1978). The drugs that were mostly prescribed were drugs for the nervous system (2004: 22.6%; 2014: 26.9%) and anti-infectives for systemic use (2004: 26.0%; 2014: 24.9%);34.3% (2004) and 39.2% (2014) of all prescriptions were off-label;62.7% of all patients received at least one off-label or unlicensed drug (2004: 70%). For 13 drugs, the licensing status changed either from off-label to label (n = 9) or vice versa (n = 4). Overall, there was no significant change neither in terms of the drugs used nor regarding their licensing status. Further studies are needed to validate these findings in a European context.

Allosteric Inhibition of SHP2 Stimulates Antitumor Immunity by Transforming the Immunosuppressive Environment.

The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.

A stepped-wedge randomized trial protocol of a community intervention for increasing lung screening through engaging primary care providers (I-STEP).

Lung cancer screening with low-dose computed tomography (LDCT) reduces lung cancer mortality, yet few eligible high-risk patients receive it annually. This protocol describes a community-partnered intervention (Toolkit) designed to support primary care practices in making referrals for lung screening and guiding patients into appropriate screening pathways. This study uses a stepped-wedge implementation design. Screening centers are randomized by readiness level to enter the intervention phase in three-month "steps" with pre-intervention data serving as the control. The primary outcome is whether delivery of the Toolkit to primary care practices results in a monthly increase in number of initial LDCT screenings. Six participating centers will identify 10 practices and reach 2-3 providers per practice to train them to use the Toolkit. The Toolkit will address known barriers to screening and referral at the patient and provider levels and provide support for required elements of screening. Toolkit components include adaptable evidence-based interventions to maximize compatibility with workflows. We hypothesize that after nine months of intervention delivery, the number of initial screening per center will double. Involving 60 practices achieves 80% power at 5% level of significance. Implementation outcomes such as adoption, acceptability, feasibility, adaptation, and sustainability will be assessed through field-notes and activity logs. LDCT for lung cancer screening currently reaches a small fraction of eligible adults. To reach the full potential to reduce mortality, primary care practices are an important venue for increasing appropriate referrals. This multidisciplinary trial will encourage acceptability and sustainability by using local knowledge and promoting partnership between providers and patients. Trial registration: ClinicalTrials.gov, NCT03958253.

The Antimalarial Natural Product Salinipostin A Identifies Essential α/ß Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites.

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/ß serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Ambulatory Monitoring of Heart Sounds via an Implanted Device Is Superior to Auscultation for Prediction of Heart Failure Events.

BACKGROUND: We compared the relationship between the third heart sound (S3) measured by an implantable cardiac device (devS3) and auscultation (ausS3) and evaluated their prognostic powers for predicting heart failure events (HFEs). METHODS AND RESULTS: In the MultiSENSE study, devS3 was measured daily with continuous values, whereas ausS3 was assessed at study visits with discrete grades. They were compared among patients with and without HFEs at baseline and against each other directly. Cox proportional hazard models were developed between follow-up visits and over the whole study. Simulations were performed on devS3 to match the limitations of auscultation. We studied 900 patients, of whom 106 patients experienced 192 HFEs. Two S3 sensing modalities correlated with each other, but at baseline, only devS3 differentiated patients with or without HFEs (P < 0.0001). The prognostic power of devS3 was superior to that of ausS3 both between follow-up visits (HR = 5.7, P < 0.0001, and 1.7, P = 0.047, respectively) and over the whole study (HR = 2.9, P < 0.0001, and 1.4, P = 0.216, respectively). Simulation results suggested this superiority may be attributed to continuous monitoring and to subaudible measuring capability. CONCLUSIONS: S3 measured by implantable cardiac devices has stronger prognostic power to predict episodes of future HFEs than that of auscultation.

RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers.

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

HeartLogic Multisensor Algorithm Identifies Patients During Periods of Significantly Increased Risk of Heart Failure Events: Results From the MultiSENSE Study.

BACKGROUND: Care of heart failure (HF) patients results in a high burden on healthcare resources, and estimating prognosis is becoming increasingly important to triage resources wisely. Natriuretic peptides are recommended prognosticators in chronic HF. Our objective was to evaluate whether a multisensor HF index and alert algorithm (HeartLogic) replaces or augments current HF risk stratification. METHODS AND RESULTS: MultiSENSE (Multisensor Chronic Evaluation in Ambulatory Heart Failure Patients) enrolled 900 patients with cardiac resynchronization therapy defibrillators enabled for collection of heart sounds, respiration, thoracic impedance, heart rate, and activity data. The HeartLogic algorithm automatically calculated a daily HF index and identified periods IN or OUT of an active alert state relative to a configurable threshold. Patients experienced 192 independently adjudicated HF events (average rate, 0.20/patient-year [pt-yr]) during 1 year of follow-up. HF event rates while IN alert was 10-fold higher than OUT of alert (0.80 versus 0.08 events/pt-yr). Combined with NT-proBNP (N-terminal pro-B-type natriuretic peptide) at enrollment (relative to 1000 pg/mL threshold, event rate was 0.42 [HIGH] versus 0.07 [LOW] events/pt-yr), substratification found the lowest risk group (LOW NT-proBNP and OUT of alert) experienced 0.02 events/pt-yr, whereas the highest risk group (HIGH NT-proBNP and IN alert) was associated with a 50-fold increased risk of an HF event (1.00 events/pt-yr) relative to the lowest risk group. CONCLUSIONS: Dynamic assessment using implantable device sensors within HeartLogic by itself or in conjunction with NT-proBNP measurements can identify time-intervals when patients are at significantly increased risk of worsening HF and potentially better triage resources to this vulnerable patient population. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov. Unique identifier: NCT01128166.

Development of an activity-based probe for acyl-protein thioesterases.

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.

Evaluation of Doxycycline-Laden Oral Bait and Topical Fipronil Delivered in a Single Bait Box to Control Ixodes scapularis (Acari: Ixodidae) and Reduce Borrelia burgdorferi and Anaplasma phagocytophilum Infection in Small Mammal Reservoirs and Host-Seeking Ticks.

A field trial was conducted on residential properties in a Lyme disease endemic area of New Jersey to determine the efficacy of Maxforce Tick Management System (TMS) bait boxes modified with doxycycline hyclate-laden bait to reduce the acarological risk of Lyme disease and the utility of galvanized steel shrouds to protect the bait boxes from squirrel depredation and ability to routinely service these devices. The strategy began with a 9-wk deployment against larvae followed by a 17-wk deployment against nymphs and larvae the second year. Passive application of fipronil reduced nymphal and larval tick burdens on small mammals by 76 and 77%, respectively, and nymphal tick abundance by 81% on treated properties. In addition, the percentage of infected small mammals recovered from intervention areas following treatment was reduced by 96% for Borrelia burgdorferi and 93% for Anaplasma phagocytophilum. Infection prevalence in host-seeking nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 93 and 61%, respectively. Results indicate that Maxforce TMS bait boxes fitted with doxycycline-impregnated bait is an effective means of reducing ticks and infection prevalence for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing Ixodes scapularis Say ticks. The protective shroud allows the device to be routinely serviced and protect against squirrel depredation.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

An in vivo multiplexed small-molecule screening platform.

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

Cysteine Cathepsin Inhibitors as Anti-Ebola Agents.

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics.

Genome-Directed Lead Discovery: Biosynthesis, Structure Elucidation, and Biological Evaluation of Two Families of Polyene Macrolactams against Trypanosoma brucei.

Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A-C from a marine actinobacterium, Micromonospora sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite Trypanosoma brucei (lobosamide A IC50 = 0.8 µM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a "molecules-to-genes-to-molecules" approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from Actinosynnema mirum. These additional analogs have allowed us to identify specific structure-activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets.

Salinipostins A-K, long-chain bicyclic phosphotriesters as a potent and selective antimalarial chemotype.

Despite significant advances in antimalarial chemotherapy over the past 30 years, development of resistance to frontline drugs remains a significant challenge that limits efforts to eradicate the disease. We now report the discovery of a new class of antimalarials, salinipostins A-K, with low nanomolar potencies and high selectivity indices against mammalian cells (salinipostin A: Plasmodium falciparum EC50 50 nM, HEK293T cytotoxicity EC50 > 50 µM). These compounds were isolated from a marine-derived Salinospora sp. bacterium and contain a bicyclic phosphotriester core structure, which is a rare motif among natural products. This scaffold differs significantly from the structures of known antimalarial compounds and represents a new lead structure for the development of therapeutic targets in malaria. Examination of the growth stage specificity of salinipostin A indicates that it exhibits growth stage-specific effects that differ from compounds that inhibit heme polymerization, while resistance selection experiments were unable to identify parasite populations that exhibited significant resistance against this compound class.

Borrelidin B: isolation, biological activity, and implications for nitrile biosynthesis.

Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.

A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics.

In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:

"Function-first" lead discovery: mode of action profiling of natural product libraries using image-based screening.

Cytological profiling is a high-content image-based screening technology that provides insight into the mode of action (MOA) for test compounds by directly measuring hundreds of phenotypic cellular features. We have extended this recently reported technology to the mechanistic characterization of unknown natural products libraries for the direct prediction of compound MOAs at the primary screening stage. By analyzing a training set of commercial compounds of known mechanism and comparing these profiles to those obtained from natural product library members, we have successfully annotated extracts based on MOA, dereplicated known compounds based on biological similarity to the training set, and identified and predicted the MOA of a unique family of iron siderophores. Coupled with traditional analytical techniques, cytological profiling provides an avenue for the creation of "function-first" approaches to natural products discovery.