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Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics. Areas covered We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) Transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement. Expert opinion LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.

A cohort consisting of asymptomatic healthcare workers donated temporal serum samples after infection with severe acute respiratory syndrome coronavirus 2. Analysis shows that all asymptomatic healthcare workers had neutralizing antibodies, that these antibodies persist for ≥60 days, and that anti-spike receptor-binding domain immunoglobulin G levels were correspondingly durable over the same time period.

As serologic assays for SARS-CoV-2 become more widely utilized, it is important to understand their performance characteristics and correlation with neutralizing antibodies. We evaluated 3 commonly used SARS-CoV-2 IgG assays (Abbott, DiaSorin, and EUROIMMUN) for clinical sensitivity, specificity, and correlation with neutralizing antibodies, and then compared antibody kinetics during the acute phase of infection.Three panels of samples were tested on every assay. Sensitivity was assessed using a panel of 35 specimens serially collected from 7 patients with RT-PCR-confirmed COVID-19. Specificity was determined using 100 sera samples collected in 2018 from healthy individuals prior to the outbreak. Analytical specificity was determined using a panel of 37 samples from individuals with respiratory illnesses other than COVID-19.Clinical sensitivity was 91.43% (95% CI 76.94-98.20%) for Abbott, and 88.57% (95% CI 73.26-96.80%) for both DiaSorin and EUROIMMUN. Clinical specificity was 99.00% (95% CI 94.55-99.97%) for Abbott and DiaSorin and 94.00% (95% CI 87.40-97.77%) for EUROIMMUN. The IgG assays demonstrated good qualitative agreement (minimum of 94%) and good correlation between the quantitative result for each combination of assays (r2 ≥ 0.90). The neutralizing antibody response did not necessarily follow the same temporal kinetics as the IgG response and did not necessarily correlate with IgG values.The 3 IgG antibody assays demonstrated comparable performance characteristics. Importantly, a qualitative positive IgG result obtained with any of the assays was associated with the presence of neutralizing antibodies; however, neutralizing antibody concentrations did not correlate well with signal to cutoff ratios.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the “divide and conquer” algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Background Because of their direct patient contact, healthcare workers (HCW) face an unprecedented risk of exposure to COVID-19. The aim of this study was to examine incidence of COVID-19 disease among asymptomatic HCW and community participants in Northern Virginia during 6 months of follow-up. Methods This is a prospective cohort study that enrolled healthy HCW and residents who never had a symptomatic COVID-19 infection prior to enrolment from the community in Northern Virginia from April to November 2020. All participants were invited to enrol in study, and they were followed at 2-, and 6-months intervals. Participants were evaluated by commercial chemiluminescence SARS-CoV-2 serology assays as part of regional health system and public health surveillance program to monitor the spread of COVID-19 disease. Findings Of a total of 1,819 asymptomatic HCW enrolled, 1,473 (96%) had data at two-months interval, and 1,323 (73%) participants had data at 6-months interval. At baseline, 21 (1.15%) were found to have prior COVID-19 exposure. At two-months interval, COVID-19 rate was 2.8% and at six months follow-up, the overall incidence rate increased to 4.8%, but was as high as 7.9% among those who belong to the youngest age group (20–29 years). Seroconversion rates in HCW were comparable to the seropositive rates in the Northern Virginia community. The overall incidence of COVID-19 in the community was 4.5%, but the estimate was higher among Hispanic ethnicity (incidence rate = 15.3%) potentially reflecting different socio-economic factors among the community participants and the HCW group. Using cross-sectional logistic regression and spatio-temporal mixed effects models, significant factors that influence the transmission rate among HCW include age, race/ethnicity, resident ZIP-code, and household exposure, but not direct patient contact. Interpretation In Northern Virginia, the seropositive rate of COVID-19 disease among HCW was comparable to that in the community.

PURPOSE OF REVIEW: We discuss recent advancements in structural biology methods for investigating sites of protein-protein interactions. We will inform readers outside the field of structural biology about techniques beyond crystallography, and how these different technologies can be utilized for drug development. RECENT FINDINGS: Advancements in cryo-electron microscopy (cryoEM) and micro-electron diffraction (microED) may change how we view atomic resolution structural biology, such that well-ordered macrocrystals of protein complexes are not required for interface identification. However, some drug discovery applications, such as lead peptide compound generation, may not require atomic resolution; mass spectrometry techniques can provide an expedited path to generation of lead compounds. New crosslinking compounds, more user-friendly data analysis, and novel protocols such as protein painting can advance drug discovery programs, even in the absence of atomic resolution structural data. Finally, artificial intelligence and machine learning methods, while never truly replacing experimental methods, may provide rational ways to stratify potential druggable regions identified with mass spectrometry into higher and lower priority candidates. SUMMARY: Electron diffraction of nanocrystals combines the benefits of both x-ray diffraction and cryoEM, and may prove to be the next generation of atomic resolution protein-protein interface identification. However, in situations such as peptide drug discovery, mass spectrometry techniques supported by advancements in computational methods will likely prove sufficient to support drug discovery efforts. In addition, these methods can be significantly faster than any crystallographic or cryoEM methods for identification of interacting regions.

The goal is to reverse cancer immune evasion at the level of the sentinel lymph node (SLN). Reduced expansion of CD8+ T cells and other innate immune effector cells in the cancer draining SLN is associated with progression and resistance to checkpoint inhibitors. Cancer-derived extracellular vesicles (EVs) that are PD-L1+ suppress immune recognition at the level of the SLN. The experimental goal is to remodel the SLN to overcome cancer EV-associated immune suppression and induce immune rejection of the tumor. We developed three methodologies for this project: a) Collection of draining lymph fluid to characterize EVs shed by 4T1 syngeneic breast tumors growing in the mammary fat pad. b) Chromatographic separation and characterization of the repertoire of EVs shed by tumors into the tumor microenvironment interstitial space using western blotting, mass spectrometry, and electron microscopy. c) Nanoparticle (NP) delivery of purified populations of EVs to the tumor draining SLN in combination with cytokine chemoattractants for innate immune cells recruitment. We characterized the in vivo interstitial fluid (IF) content of a GFP-4T1 syngeneic murine cancer model to study resident IF EVs transit to the draining lymph node. GFP labeling confirmed the IF EV tumor cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; including PINK1 and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial pinching), and VPS35, SEC22b, and Rab33b (vacuolar sorting). SLN immune cell populations could be massively remodeled by introducing hydrogel NPs which have a controlled release of T-cell and dendritic cell chemoattractant to the subcapsular sinus. NPs were successfully used to deliver concentrated packages of EVs subpopulations to the SLN. Introduction of the large CD81-/VEGF+/PD-L1- EV subpopulation (amphisome characteristics) to the SLN augmented tumor growth, angiogenesis, and metastasis, even when cytokine induction was used to remodel the SLN. In marked contrast, introduction of the CD81+/PD-L1+ EV subpopulation (containing mitophagy components) to the SLN in combination with NP release of chemoattractants, induced immune rejection of the syngeneic breast cancer, reducing tumor growth, and blocking metastasis. These findings demonstrate that different populations of EVs have opposite effects on cancer immune evasion at the level of the SLN and that EV-mediated immune suppression can be reversed by SLN remodeling to augment dendritic and CD8+ T cells. Citation Format: Marissa Howard, James Erickson, Amanda Haymond, Alessandra Luchini, Fatah Kashanchi, Lance Liotta. Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3516.

Ovarian cancer is the deadliest cancer of the female reproductive system, with the majority of patients experiencing relapse on first-line platinum-based chemotherapies. Identifying potential targets for additional second-line therapies is critical for treating platinum-resistant ovarian cancer. To that end, the evolution of platinum resistance in a cell-based model of ovarian cancer was monitored via characterization of proteomic changes in cancer-derived extracellular vesicles (EVs) after progressive rounds of cisplatin treatment. The content of cancer-derived EVs was analyzed via reverse phase protein array, mass spectrometry, and Western blotting to identify pathways that could be targeted by second-line therapies. Several therapeutically relevant markers were upregulated, including phospho-EGFR (Y1068), multidrug transporter ABCE1, and PD-L1. Monoclonal antibody-based therapies (mAbs) targeting PD-1/PD-L1 have been deployed successfully as second-line treatments for a variety of cancers, but their success has yet to be translated to ovarian cancer. Current mAb-based immunotherapies suffer from several pitfalls including poor tissue penetration, a long-standing issue in ovarian cancer treatment. To offer an alternative to mAb-based immunotherapies, we developed a small interfering peptide consisting of a portion of the PD-L1 interface sequence. This lead peptide underwent several rounds of in silico optimization via molecular dynamics and modeling simulations to identify modifications that could improve affinity for PD-1 or stability in serum. When efficacy was assessed in vitro via Amplified Luminescent Proximity Homogeneous Assay screening, the modifications to the lead peptide were not shown to improve efficacy. However, serum stability studies indicated the modifications dramatically improved peptide stability, with detectable levels still quantifiable via mass spectrometry after 48 hours. The stabilized peptide, titled MN1.4, was chosen for further efficacy testing in cell-based models. A cisplatin-treated daughter line from the OVCAR8 high grade serous ovarian carcinoma cell line was created to model the effects of clinical platinum-based chemotherapy treatments on the most common ovarian cancer subtype. OVCAR8 cells were co-cultured in the presence of stimulated Jurkat T cells to measure markers of T cell activation, including IL-2, via ELISA after treatment with MN1.4 in the presence or absence of EVs derived from cisplatin-treated cells. This model allows for examination of the potential sensitization effects of first-line cisplatin treatment on PD-1/PD-L1 immunotherapy, and how cancer-derived EVs from cisplatin-treated cells can abrogate this effect. We conclude that exosomal PD-L1 may impair responses to PD-1/PD-L1 immunotherapy and should be considered when assessing the viability of this treatment option for ovarian cancer patients. Citation Format: Rachel Carter, Marissa Howard, James Erickson, Paul Russo, Alessandra Luchini, Amanda Haymond, Fatah Kashanchi, Mikell Paige, Lance Liotta. Investigating PD-1/PD-L1 checkpoint inhibition as a treatment for platinum-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1117.

Immunotherapy has shown tremendous promise in the treatment of blood cancers; however, treatment typically fails when attempted with solid tumors, such as breast cancer. However, the cases where immunotherapies have been successful in breast cancer, the results have been remarkable: full remission of stage 4 disease has been observed. This highlights the need to elucidate how the tumor microenvironment (TME) fosters a context of molecular crosstalk that is hostile to inflammatory, anti-tumor immunological responses that present a significant roadblock to consistently successful immunotherapy. Aberrant IL-33 present in the TME is believed to be one of these major factors that drives tumor progression via its ligand, ST2, and the subsequent recruitment of IL1RAcP. This activates MyD88 cascades in dysfunctional myeloid immune cells and myeloid derived suppressor cells (MDSCs), eliciting immunosuppression that promotes tumor tolerance and subsequent disease progression. To this end, preventing IL-33/ST2 signaling activation is an attractive target to possibly manipulate immune cells to recognize and mount a response against tumor cells. Our previous work has identified 4 IL1RAcP hotspot residues of interaction between it and the ST2/IL-33 complex: R157, K238, K343, and K346. Here, we designed peptides, which mimic these hotspots and the adjacent residues that interact with the ST2/IL-33 complex. Wild type peptides inhibit trimer formation in vitro as determined by immunoprecipitation and chemical crosslinking experiments. Modifications introduced to increase rigidity and stability increased affinity in vitro and showed inhibition of downstream signaling in reporter gene cell models. Citation Format: Jessica Roman, Rachel Carter, Alessandra Luchini, Lance Liotta, Remi Veneziano, Amanda Haymond. New hybrid molecular modalities comprised of DNA-origami and interfering peptides as inhibitors of protein-protein interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3545.

The Substance Abuse and Mental Health Services Administration (SAMHSA) recently authorized oral fluid (OF) as a preferable biofluid for drugs of abuse (DOA) screening compared to urine, and they required that each screening method be confirmed by a laboratory test. We developed a DOA mass spectrometry (MS) assay optimized for undiluted OF as a matching confirmatory test for the EZ-Saliva point of care (POC), split sample, rapid visual test. Using a double isotope ratio standardization, we achieved a limit of detection of 2 = 0.99, lack of interference (90%). Stability from degradation exceeded 72 hours. The lateral flow immunoassay strips of the POC exhibited a dose-dependent response, with a 90% sensitivity and 100% specificity for N=22 self-reported, THC patient OF, digitized for quantitation. We conclude that the split sample POC device in combination with the MS assay meets the SAMHSA stated requirements for a POC test with a laboratory confirmation. Split sample collection has significant advantages because it minimizes potential error created by taking a separate OF sample for laboratory confirmation. We recommend scaling to a larger validation study set and quantification of user OF THC levels that correlate with driver impairment levels.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as ‘simultaneous multifiber headspace solid-phase microextraction’ (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,β-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC(50) = 13 nM) without significant inhibition of HepG2 cells (IC(50) > 50 μM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.

Introduction: Signal transduction cascades drive cellular proliferation, apoptosis, immune, and survival pathways. Proteins have emerged as actionable drug targets because they are often dysregulated in cancer, due to underlying genetic mutations, or dysregulated signaling pathways. Cancer drug development relies on proteomic technologies to identify potential biomarkers, mechanisms-of-action, and to identify protein binding hot spots. Areas covered: Brief summaries of proteomic technologies for drug discovery include mass spectrometry, reverse phase protein arrays, chemoproteomics, and fragment based screening. Protein-protein interface mapping is presented as a promising method for peptide therapeutic development. The topic of biosimilar therapeutics is presented as an opportunity to apply proteomic technologies to this new class of cancer drug. Expert opinion: Proteomic technologies are indispensable for drug discovery. A suite of technologies including mass spectrometry, reverse phase protein arrays, and protein-protein interaction mapping provide complimentary information for drug development. These assays have matured into well controlled, robust technologies. Recent regulatory approval of biosimilar therapeutics provides another opportunity to decipher the molecular nuances of their unique mechanisms of action. The ability to identify previously hidden protein hot spots is expanding the gamut of potential drug targets. Proteomic profiling permits lead compound evaluation beyond the one drug, one target paradigm.

Protein–protein interactions lie at the heart of many biological processes and therefore represent promising drug targets. Despite this opportunity, identification of protein–protein interfaces remains challenging. We have previously developed a method that relies on coating protein surfaces with small-molecule dyes to discriminate between solvent-accessible protein surfaces and hidden interface regions. Dye-bound, solvent-accessible protein regions resist trypsin digestion, whereas hidden interface regions are revealed by denaturation and sequenced by MS. The small-molecule dyes bind promiscuously and with high affinity, but their binding mechanism is unknown. Here, we report on the optimization of a novel dye probe used in protein painting, Fast Blue B + naphthionic acid, and show that its affinity for proteins strongly depends on hydrophobic moieties that we call here “hydrophobic clamps.” We demonstrate the utility of this probe by sequencing the protein–protein interaction regions between the Hippo pathway protein Yes-associated protein 2 (YAP2) and tight junction protein 1 (TJP1 or ZO-1), uncovering interactions via the known binding domain as well as ZO-1's MAGUK domain and YAP's N-terminal proline-rich domain. Additionally, we demonstrate how residues predicted by protein painting are present exclusively in the complex interface and how these residues may guide the development of peptide inhibitors using a case study of programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1). Inhibitors designed around the PD-1/PD-L1 interface regions identified via protein painting effectively disrupted complex formation, with the most potent inhibitor having an IC(50) of 5 μm.

Blocking the 2-C-methyl-D-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of 2. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth. (Figure Presented).

Breast cancer subtypes are currently defined by a combination of morphologic, genomic, and proteomic characteristics. These subtypes provide a molecular portrait of the tumor that aids diagnosis, prognosis, and treatment escalation/de-escalation options. Gene expression signatures describing intrinsic breast cancer subtypes for predicting risk of recurrence have been rapidly adopted in the clinic. Despite the use of subtype classifications, many patients develop drug resistance, breast cancer recurrence, or therapy failure. Areas covered: This review provides a summary of immunohistochemistry, reverse phase protein array, mass spectrometry, and integrative studies that are revealing differences in biological functions within and between breast cancer subtypes. We conclude with a discussion of rigor and reproducibility for proteomic-based biomarker discovery. Expert commentary: Innovations in proteomics, including implementation of assay guidelines and standards, are facilitating refinement of breast cancer subtypes. Proteomic and phosphoproteomic information distinguish biologically functional subtypes, are predictive of recurrence, and indicate likelihood of drug resistance. Actionable, activated signal transduction pathways can now be quantified and characterized. Proteomic biomarker validation in large, well-designed studies should become a public health priority to capitalize on the wealth of information gleaned from the proteome.

The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

Protein-protein interactions are thought to be the next frontier in drug discovery. However, there are several well-known challenges facing development of protein-protein interaction (PPI) inhibitors that lead to slow development in the field, including difficulty identifying PPIs and difficulty designing small molecule inhibitors of relatively flat, featureless PPIs. We address these difficulties with the development of a novel method of discovering PPI hotspots, called protein painting. This technique relies on non-covalent labeling of solvent- accessible protein surfaces using small molecular dyes optimized for protein binding. The dyes block access to trypsin cleavage sites, allowing for digestion only of undyed interface regions following denaturation of the protein complex. Interface regions can be identified using mass spectrometry and used as target sequences for drug development. Here we introduce new dye chemistries and elucidate their mechanism of protein binding for the first time. This allows for rapid identification of functionally-relevant hotspots without the need to screen many dye chemistries to optimize surface coverage of the protein complex. We applied this method to elucidate functional hotspots for immmuno-oncology targets PD-1 and PD-L1 and Hippo pathway targets YAP2 and tight junction protein ZO-1. To further functionally validate the hotspot regions identified, we focused on the case study of PD-1 and PD-L1. We discovered a hotspot of PD-1 Lys 78 in the protein-protein interface, and rationally designed a series of 8 peptide inhibitors to target this hotspot. The most active peptide YRCMISYGGADYKRITV derived from PD-L1 disrupted the PD-1/PD-L1 complex with an IC50 of 5.07 µM. The predicted binding site of this peptide on PD-1 overlaps the binding site of therapeutic anti-PD-1 antibody pembrolizumab; crystal structures of pembrolizumab and PD-1 show hydrogen bonding between the antibody and our identified hotspot Lys 78. Furthermore, we prepared a cyclized analog peptide CYRAMISYGGADYKRITC by disulfide bond stapling to increase peptide stability and found that this did not significantly reduce inhibitor potency, with an IC50 of 8.02 µM. Taken together, this data suggests a specific region of PD-1 found within the larger PD-1/PD-L1 interface that may serve as a target for development of next generation small molecule PD-1/PD-L1 inhibitors. By focusing drug discovery efforts against only the PPI hotspot regions, we may accelerate drug development against these difficult targets. Citation Format: Amanda Still, Douglass Dey, Rachel Carter, Angela Dailing, Mikell Paige, Lance Liotta, Alessandra Luchini. Functionally important hotspot interfaces between immune-oncology targets PD-1 and PD-L1 and between Hippo pathway targets YAP2 and tight junction protein ZO-1 are identified using a protein-protein interaction technique optimized with novel dye chemistries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 982.

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or “black death,” both rely on the MEP pathway for isoprene production. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group, and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homolog, with the best analogs displaying nM IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and diPOM prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3–12 µg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologs from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.

In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents.

In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 μM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents.