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).

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.

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.

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.

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.

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.

Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products.

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

Precipitation and temperature as predictors of the local abundance of Ixodes scapularis (Acari: Ixodidae) nymphs.

Populations of Ixodes scapularis Say nymphs were surveyed at a Lyme disease- endemic area for 8 consecutive yr (1998-2005) to characterize annual changes in abundance. Precipitation and temperature were also monitored over the period 1998-2004 to determine their potential value as predictors of tick abundance. Although both parameters showed annual variation, no statistical differences in the annual abundance of I. scapularis nymphs were observed over the 8-yr period. Our results suggest that precipitation and temperature were not predictive of the abundance of I. scapularis nymphs.

Effectiveness of the 4-Poster passive topical treatment device in the control of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in New Jersey.

Twenty-five "4-Poster" feeders were placed throughout a 5.2 km(2) study area within a secured military facility situated in a hyperendemic area for Lyme disease in central Monmouth County, New Jersey. Calculated levels of control, relative to untreated areas, peaked at 82.7%, 77.3%, and 94.2% for of host-seeking Ixodes scapularis Say larvae, nymphs, and adults, respectively, within 5 years of deployment. Control of host-seeking Amblyomma americanum (L.) peaked at 99.2%, 89.5%, and 96.9% for larvae, nymphs, and adults, respectively, during the treatment period. Tick burdens on hunter-killed deer were significantly reduced on deer harvested from the treatment area and on deer that had consumed bait corn. Populations of subadult I. scapularis and A. americanum demonstrated some rebound effect following the removal of 4-Posters, but treatment area tick populations remained lower than control area populations 2 years following withdrawal of the 4-Posters. However, control of I. scapularis adults declined to 20.7% by the third fall activity period following removal of the 4-Posters. The posttreatment phase of the study was of insufficient duration to evaluate continued population rebound of adults and subadults during subsequent activity periods.

Ability of two natural products, nootkatone and carvacrol, to suppress Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey.

We evaluated the ability of the natural, plant-derived acaricides nootkatone and carvacrol to suppress Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae). Aqueous formulations of 1 and 5% nootkatone applied by backpack sprayer to the forest litter layer completely suppressed I. scapularis nymphs through 2 d. Thereafter, the level of reduction gradually declined to < or =50% at 28 d postapplication. Against A. americanum nymphs, 1% nootkatone was less effective, but at a 5% concentration, the level of control was similar or greater to that observed with I. scapularis through 21 d postapplication. Initial applications of 0.05% carvacrol were ineffective, but a 5% carvacrol formulation completely suppressed nymphs of both species through 2 d and resulted in significant reduction in I. scapularis and A. americanum nymphs through 28 and 14 d postapplication, respectively. Backpack sprayer applications of 5% nootkatone to the shrub and litter layers resulted in 100% control of I. scapularis adults through 6 d, but the level of reduction declined to 71.5% at 28 d postapplication. By contrast, high-pressure applications of 2% nootkatone to the litter layer resulted in 96.2-100% suppression of both I. scapularis and A. americanum nymphs through 42 d, whereas much lower control was obtained from the same formulation applied by backpack sprayer. Backpack sprayer application of a 3.1% nootkatone nanoemulsion resulted in 97.5-98.9 and 99.3-100% reduction in I. scapularis and A. americanum nymphs, respectively, at 1 d postapplication. Between 7 d and 35 d postapplication, the level of control varied between 57.1% and 92.5% for I. scapularis and between 78.5 and 97.1% for A. americanum nymphs. The ability of natural products to quickly suppress and maintain significant control of populations of these medically important ticks at relatively low concentrations may represent a future alternative to the use of conventional synthetic acaricides.