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KRAS is one of the most frequently mutated genes in cancer and was long considered undruggable until the recent discovery of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most clinically advanced of these first-generation molecules have demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients fail to achieve a clinical response and acquired resistance is common. Resistance to first-generation inhibitors can be driven by upregulation of the activated (GTP-bound) form of KRASG12C, which remains an undrugged form of the oncoprotein. Here we report the discovery of FMC-376, a novel inhibitor of the activated, GTP-bound, form of KRASG12C, which also potently inhibits the inactive, (GDP-bound), form of KRASG12C. FMC-376 was discovered through the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery. FMC-376 binds KRAS in the switch II pocket, rapidly forming a covalent bond with cysteine 12 in the presence of either GDP or GTP. X-ray crystallography demonstrated that Cys12 adopts a novel confirmation in forming a covalent bond with FMC-376. This results in potent inhibition of RAF1 and PI3Kα effector interactions (IC50 = 0.007 μM for both respectively at 2 h) in contrast to sotorasib or adagrasib (IC50 > 50 and ~ 5 μM respectively). FMC-376 treatment results in potent anti-tumor activity across a panel of KRASG12C mutant tumor cell lines, sparing non- KRASG12C cell lines. To model resistance mediated by activated KRASG12C, a mutation that abrogates GTPase activity (A59G) was introduced into KRASG12C. This upregulation of GTP-bound KRASG12C drives significant (>10-fold) resistance to both adagrasib and sotorasib in tumor cell viability assays whereas FMC-376 remains equipotent in settings where GTP-bound KRASG12C is upregulated. Evaluation of FMC-376 in models where EGFR signaling (a suspected mechanism of clinical resistance) is induced demonstrated rapid and durable target engagement in contrast to both sotorasib and adagrasib which show decreased effectiveness after EGF stimulation. Further evaluation of FMC-376 in vivo has demonstrated rapid and durable KRASG12C target occupancy (>90%) and pathway inhibition in tumors, resulting in regression of CDX/PDX tumor models. FMC-376, an inhibitor of both active and inactive forms of KRASG12C, provides a differentiated mechanism of action with the potential for broader and more durable response in the clinic. Citation Format: Snahel Patel, Barun Bhhatarai, Philamer Calses, Daniel Erlanson, Robert Everley, Susan Fong, Phil Gerken, Johannes C. Hermann, Tiep Le, Li-kai Liu, Evan McMahon, Richard M. Neve, Tony Phan, Allison Roberts, Mikayla Shanafelt, Sophie Siemsgluess, Jocelyn Staunton, Yan Wang, Weiru Wang, Monika Williams, Kevin R. Webster. Discovery of FMC-376 a novel orally bioavailable inhibitor of activated KRASG12C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1142.

KRAS is one of the most frequently mutated genes in cancer with alterations occurring in > 14% of all tumors. Recent advances have led to the discovery and development of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most advanced of these first-generation molecules demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients failed to achieve a clinical response and acquired resistance can be rapid. One hypothesis to explain tumor resistance is the failure of existing inhibitors to recognize the activated (GTP-bound) form of KRASG12C that can be upregulated in response to these first-generation inhibitors. Here we report the discovery of a series of novel inhibitors that effectively inhibit both the GTP- and GDP-bound forms of KRASG12C. These “dual-acting” inhibitors bind in the switch II pocket of both GTP-bound and GDP-bound KRASG12C and rapidly form a covalent bond with cysteine 12. This results in significantly increased inhibition of RAF1 and PI3Kα effector interactions (IC50 < 5 nM at 2 hrs.) in comparison to inactive state inhibitors. Dual targeting of both GTP- and GDP-bound KRASG12C results in potent cellular activity in models that are both sensitive (NCI-H358 and MIA PaCa-2) and resistant (NCI-H2122) to adagrasib and sotorasib. In contrast to adagrasib and sotorasib which are less effective in the NCI-H2122 cell line model, dual-acting inhibitors of GTP- and GDP-bound KRASG12C elicit rapid inhibition of pERK in < 1 hour with sustained inhibition of MAPK signaling through 48 hours. To model resistance to first generation inhibitors, an A59G mutation was introduced into KRASG12C, abrogating GTPase activity. This decreases the activity of both adagrasib and sotorasib in tumor cell viability assays by more than an order of magnitude whereas dual-acting inhibitors of GTP- and GDP-bound KRASG12C are equally effective in the G12C/A59G and parental G12C cell lines. Evaluation of dual-acting inhibitors of KRASG12C in vivo demonstrated rapid and > 90% KRASG12C target occupancy, resulting in regression of MIA PaCa-2 tumors. Dual-acting inhibitors of both the active and inactive states of KRASG12C may provide the potential for broader and more durable responses in the clinic. Citation Format: Philamer Calses, Sam Clark, Jacob Corpuz, Susan Fong, Phil Gerkin, Mohammad Hekmatnejad, Evan McMahon, Megan Murray, Truc Nguyen, Tony Phan, Allison Roberts, Phillip Schwartz, Mikayla Shanafelt, Hiroko Tanaka, Jennifer Tomczyk, John Widen, Monika Williams, John Eksterowicz, Daniel Erlanson, Marie Evangelista, Johannes Hermann, Richard M. Neve, Snahel Patel, Kevin R. Webster. Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein [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 3601.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.

In pursuit of effective therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen amino acid peptide binds to major structural protein VP1 in a new location within the pore with a low nanomolarKD. Alanine scanning of the peptide identified three key residues, substitution of each of which results in ∼1000-fold loss of affinity with a concomitant reduction in antiviral activity. NMR spectroscopy and an X-ray structurally-guided model demonstrate specific binding of the peptide to the pore of the VP1 pentamer that constitutes the BKV capsid. Cell-based assays with the peptide demonstrate nanomolar inhibition of BKV infection and suggest that the peptide likely blocks the viral entry pathway between endocytosis and escape from the host cell ER. The peptide motif is highly conserved among the polyomavirus clade, and homologous peptides exhibit similar binding properties for JC polyomavirus and inhibit infection with similar potency to BKV in a model cell line. Substitutions within VP1 or VP2/3 residues involved in VP1-peptide interaction negatively impact viral infectivity, potentially indicating the peptide-binding site within the VP1 pore is relevant for VP1-VP2/3 interactions. The inhibitory potential of the peptide-binding site first reported here may present a novel target for development of new anti-polyomavirus therapies. In summary, we present the first anti-polyomavirus inhibitor that acts via a novel mechanism of action by specifically targeting the pore of VP1.

The success of hit-finding campaigns relies on many factors, including the quality and diversity of the set of compounds that is selected for screening. This paper presents a generalized workflow that guides compound selections from large compound archives with opportunities to bias the selections with available knowledge in order to improve hit quality while still effectively sampling the accessible chemical space. An optional flag in the workflow supports an explicit complement design function where diversity selections complement a given core set of compounds. Results from three project applications as well as a literature case study exemplify the effectiveness of the approach, which is available as a KNIME workflow named Biased Complement Diversity (BCD).

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery., PLoS ONE, 12 (4), ISSN:1932-6203

A series of arylaminobenzimidazoles was designed and synthesized as Raf kinase inhibitors. Exploration of the structure-activity relationship resulted in compounds that are potent in vitro and show desirable in vivo properties.

Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug. We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate and measured directly the enzymatically released acetate. Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM. We now report the synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

Utilizing structure-based drug design, a novel dihydropyridopyrimidinone series which exhibited potent Hsp90 inhibition, good pharmacokinetics upon oral administration, and an excellent pharmacokinetic/pharmacodynamic relationship in vivo was developed from a commercial hit. The exploration of this series led to the selection of NVP-HSP990 as a development candidate.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

A general strategy has been developed for rapidly identifying peptides that alter cellular function, which has revealed a series of peptides that inhibit platelet aggregation. First whole cells—platelets in this case—were used as an affinity matrix to isolate cell-binding phage from a phage-display, random-peptide library. In this step 3 × 107 different filamentous phage, each encoding and displaying a different random peptide, were screened as a single pool. Since it is the peptide displayed on the surface of the phage that presumably causes a particular phage to bind specifically to a cell, synthetic peptides were then made based on the predicted sequences of the displayed peptides. Because the initial affinity selection step had focused attention on a handful of peptides, each of the 17 synthetic peptides could be individually tested in an appropriate functional assay, such as platelet aggregation. The majority of the 17 different peptides that we analyzed inhibited platelet aggregation; these peptides could be grouped into at least five different motifs including the expected RGD motif. It is believed that this strategy, which requires neither purification nor prior knowledge of a particular target receptor, will prove to be generally useful for identifying peptide ligands that influence cellular function. © 1994 Wiley-Less, Inc.

The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.

Mutated forms of KRAS are no longer able to switch effectors between “on” and “off” states. It is known that the function of KRAS is controlled by key parts in the C-terminus, including six consecutive lysines, a terminal prenyl moiety and a terminal carboxymethyl functional group. We set out to discover compounds which would inhibit the function of mutated KRAS as an activator for effectors. This campaign yielded several compounds that blocked biochemical and cellular functions of KRAS with low micromolar activity while not affecting markers outside of KRAS pathways in cells. In order to understand the mode of binding of these compounds to KRAS, we generated different forms of the protein, including unprenylated truncated and fully processed full-length protein. NMR studies with truncated protein (amino acids 1-169) identified a site at which compound binding stabilized the inactive conformation of KRAS. This site is located adjacent to switch-II and is similar to sites described by others. The Kd determined for this binding event is almost 3 orders of magnitude higher than the IC50 and EC50 values measured in biochemical and cellular assays. In order to understand this difference, we developed a biophysical assay using the Fortebio system which enabled binding studies in a system with full-length prenylated protein in the presence of lipids, to match the context of the biochemical and cellular assays. Micromolar binding to the full-length prenylated KRAS protein was observed in the Fortebio assay and binding was not observed in the absence of prenylation, consistent with the near millimolar Kd observed by NMR for truncated KRAS. Curiously, similar micromolar binding was seen to a peptide derived from the C-terminus of KRAS (amino acids 168-185) with and without prenyl modification while related compounds that do not bind to the full-length prenylated KRAS also do not bind to these peptides. It is still unclear whether binding to the terminal peptide in lipid context is related to the binding site adjacent to switch-II. From a drug discovery perspective, it remains to be confirmed whether current inhibitors can be optimized. Citation Format: Johanna Jansen, Wolfgang Jahnke, Susan Fong, Laura Tandeske, Charles Wartchow, Keith Pfister, Tatiana Zavorotinskaya, Anke Blechschmidt, Dirksen Bussiere, Yumin Dai, Jeff Dove, Eric Fang, David Farley, Jean-Michel Florent, John Fuller, Simona Gokhin, Alvar Gossert, Mohammad Hekmat-Nejad, Chrystèle Henry, Julia Klopp, Bill Lenahan, Andreas Lingel, Arndt Meyer, Jamie Narberes, Gwynn Pardee, C Gregory Paris, Savithri Ramurthy, Paul Renhowe, Sebastien Rieffel, Kevin Shoemaker, Sharadha Subramanian, Tiffany Tsang, Stephania Widger, Armin Widmer, Isabel Zaror, Stephen Hardy. Inhibiting mutated KRAS, a broken switch of effector pathways. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B38. doi: 10.1158/1557-3125.RASONC14-B38

BEHAVIOR IN RATS? SUSAN FONG, PETER VAN EERDEN, ILONA VATHY, Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, New York, University of South Dakota School of Medicine, Obstetrics and Gynecology, Sioux Falls, South Dakota, Albert Einstein College of Medicine, Department of Psychiatry and Behavioral Sciences, Bronx, New York OBJECTIVE: To determine whether prenatal stress in the form of subcutaneous injections adversely affects maternal behaviors in rats, and whether morphine can decrease these effects. STUDY DESIGN: Pregnant rats at gestational day 11 were randomly assigned to treatment with saline (n = 8), morphine (n = 7), or neither (n = 7). Saline or morphine was given subcutaneously every 12 hours on gestational days 11 to 18. Litters were cross fostered upon delivery. Rats were monitored on postnatal days 2 to 16 for retrieval of the pups. Prior to this test pups were removed from home cages for 5 minutes, then replaced in the home cages distal to the nest, and time to retrieval of first and all pups monitored. The postnatal day at which retrievals took longer than 10 minutes was recorded. Postnatal days 2 to 22, rats were monitored 10 times daily for non-maternal behaviors. RESULTS: Using the log-rank test, there was a significant difference (p = 0.012) between the saline-exposed group and the morphine-exposed and control groups in the postnatal day at which time to retrieval of the first pup occured at greater than 10 minutes (saline 14.4 +/ 1.9, morphine 9.8 +/ 2.6, control 9.8 +/ 2.4); however there was no difference between groups in the postnatal day at which retrieval of all pups in the litter occured at greater than 10 minutes (p = 0.16). Using one way ANOVA, the mean occurence of non-maternal behaviors (saline 29.2 +/ 10.4, morphine 86.0 +/ 19.6, control 81.8 +/ 26.5) was different between the three groups (F = 19.9, p!0.0001). CONCLUSION: Prenatal exposure to stress in the form of subcutaneous injections resulted in an increased occurance and duration of maternal behaviors towards their offspring as compared to controls. The addition of morphine to the stress was associated with occurance and duration of maternal behavior identical to that of controls.

Further application of the tritiated amino acid suicide selection procedure, designed to select conditional protein synthesis mutants, has yielded over 300 aminoacyl-tRNA synthetase mutants of Chinese hamster ovary cells, representing seven complementation groups. A number of these mutants and their revertants were used to examine the extent of X-chromosome linkage or other functional hemizygosity in CHO cells. Our results indicated that none of the seven different aaRSloci tested was X linked, and the the glnRSlocus, unlike the asnRSand leuRSloci, does not appear to be functionally hemizygous in CHO cells.

Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 2.4.2.8) and APRT (adenine phosphoribosyltransferase EC 2.4.2.7) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 microgram/ml of azaadenine, 14/16 of the resistant clones picked and tested had approximately 1/2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 microgram/ml azaadenine. Most of the highly resistant colonies isolated (21/24) had very low in vitro APRT activity. The optimal conditions for detection of TGr and AAr mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AAr and by 4-5 days for TGr. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10(5) to 1 x 10(6) cells per 100-mm petri dish (i.e. 1.6 x 10(3) to 1.6 x 10(4) cells/cm2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AAr, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.

Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2(125) (rIL 2(125)). The rIL 2(125) used in these experiments was purified to homogeneity from Escherichia coli, contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4 X 10(6) units/mg. Human rIL 2(125) was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species. Long-term cultures were highly sensitive to low levels of rIL 2(125) and showed dose-dependent responses when used in short-term IL 2 assays. Bovine and porcine PBMC preincubated with human rIL 2(125) for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.