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The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.

In vivo analyses using an autonomously replicating Agrobacterium/geminivirus vector have enabled identification of AU-rich intronic elements critical for 5' and 3' splice site selection in dicot plant nuclei and development of a model for pre-mRNA intron recognition in plant nuclei. To determine the minimal length, spacing and nucleotide compositions constraining recognition of the 3' boundary of an intron, two or four nucleotide substitutions have been introduced into the two AU-rich elements located between 50 and 66 nucleotides upstream from the 3' splice site of maize Adh1 intron 3. In each case tested, substitutions in the distal left element (-62 to -66) inactivate the downstream 3' splice site at -1 more effectively than substitutions in the proximal right element (-50 to -55). Guanosine or cytosine substitutions in either element reduce recognition of the -1 site significantly; adenosine substitutions have a less severe effect. Mutations in both of these AU elements additively block recognition of the downstream 3' splice site. The strong additive effect of these mutations supports a model in which short sets of AU islands bind interactive factors and cooperatively modulate usage of the downstream splice site. In contrast to the uridine requirements documented for the 3' terminus of plant introns, adenosines are partially interchangeable with uridines within this internal region of the intron.

The MAPK signaling cascade, comprised of the RAS GTPases, the RAF, MEK1/2 and ERK1/2 kinases is frequently deregulated in cancer. ERK1 and ERK2 transmit signals generated by mutant BRAF, Ras or by activated receptor tyrosine kinases to a wide range of nuclear and cytoplasmic substrates, resulting in signal amplification, cell growth, migration and survival. ERK1 and ERK2 have been considered as redundant because of their high homology, large number of overlapping substrates, and ability to substitute for each other in genetically engineered mouse models. Nevertheless, several investigators have identified non-redundant roles for ERK isoforms in oncogenesis; for instance, ERK2, but not ERK1, appears to be responsible for RASmut induced epithelial-to-mesenchymal transformation. Besides, each of the ERK isoforms employs spatially distinct substrate docking domains, DEF (docking site for ERK FXFP) and D (docking domain), to signal to different subsets of substrates and differentially transmit signals downstream. We set out to determine the roles of ERK isoforms as well as DEF- and D-domain dependent signaling in the survival of melanoma tumor cells expressing activating BRAF mutations which are highly sensitive to pharmacological inhibitors of RAF, MEK1/2 and ERK1/2. We designed ERK1 and ERK2 mutants resistant to ATP-competitive ERK1/2 inhibitors and employed auto-activating, MEK-independent, ERK1 and ERK2 mutants to ask if BRAFmut melanoma survival is dependent on either or both ERK isoforms. In addition, we used RNAi and zinc-finger nucleases’ to knockdown or delete each of ERK isoforms. These experimental approaches consistently demonstrated that ERK2, but not ERK1, was the sole driver of cell survival in multiple BRAFmut melanoma cell lines. Moreover, genome-wide gene expression analysis indicated that ERK2, but not ERK1, was largely responsible for transcriptional effects imposed by pharmacological RAF, MEK1/2 or ERK1/2 inhibitors. Thus, in BRAFmut melanoma, functions of ERK1 and 2 are not redundant, and ERK1 cannot substitute for a disabled ERK2. Next, we introduced DEF- and D-substrate docking domain mutations into an ERK inhibitor resistant ERK2 to investigate whether signaling through either domain is sufficient to support melanoma survival. We observed that signaling through D- or DEF- domains of ERK2 had differential effects on gene expression and substrate phosphorylation. Consequently, we have found that a subset of melanoma cell lines was sensitive to elimination of DEF- docking domain interactions, whereas another subset of cell lines tolerated mutations in the DEF-site. Interactions and signaling through ERK D-docking site were dispensable for survival of all melanoma cell lines tested. These data suggest potential novel approaches to target oncogenic MAPK pathway. Citation Format: Tatiana Zavorotinskaya, Upasana Mehra, Yumin Dai, Michel Faure, Ken Crawford, Karen Yu, Jan Marie Cheng, Xiaolei Ma, Jan Xuan, Kelly Yan, Mohammad Hekmat-Nejad, Hanne Merritt, Darrin Stuart, Charles Voliva. Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-121. doi:10.1158/1538-7445.AM2014-LB-121

We determined the extent to which additional binding energy could be achieved by diversifying the complementarity determining regions (CDRs) located in the center of the antibody combining site of C6.5, a human single-chain Fv (scFv) isolated from a non-immune phage library which binds the tumor antigen c-erbB-2. CDR3 of the light (V(L)) and heavy (V(H)) chain variable region of C6.5 were sequentially mutated, the mutant scFv displayed on phage, and higher affinity mutants selected on antigen. Mutation of V(L) CDR3 yielded a scFv (C6ML3-9) with a 16-fold lower Kd (1.0 x 10(-9) M) than C6.5. Due to its length of 20 amino acids, four V(H) CDR3 libraries of C6ML3-9 were constructed. The greatest increase in affinity from a single library was ninefold (Kd = 1.1 x 10(-10) M). Combination of mutations isolated from separate V(H) CDR3 libraries yielded additional ninefold decreases in Kd, resulting in a scFv with a 1230-fold increase in affinity from wild-type C6.5 (Kd = 1.3 x 10(-11) M). The increase in affinity, and its absolute value, are comparable to the largest values observed for antibody affinity maturation in vivo or in vitro and indicate that mutation of V(L) and V(H) CDR3 may be a particularly efficient means to increase antibody affinity. This result, combined with the location of amino acid conservation and substitution, suggests an overall strategy for in vitro antibody affinity maturation. In addition, the affinities and binding kinetics of the single-chain Fv provide reagents with potential tumor targeting abilities not previously available.

Genetic alterations in the Ras/Raf/MEK/ERK pathway are among the most common in human cancers. Up to 70% of melanomas harbor B-Raf mutations, and roughly 90% of pancreatic tumors have K-Ras mutations. To address these Raf pathway-driven cancers, small molecule Raf kinase inhibitors have been developed and are currently under clinical investigation. In B-RafV600E cells, Raf compounds inhibit signaling through MEK and ERK, resulting in the expected anti-proliferative effects. Paradoxically, in wild-type Raf cells and in mutant Ras cells, these compounds induce downstream signaling and can induce cell growth in some settings in vitro. While the induction of downstream signaling has previously been attributed to published Raf pathway feedback loops, this has not been proven directly. In fact, we show here that induction of pMEK and pERK can occur within minutes of Raf compound treatment, even before reported feedback phosphorylation events are seen on B-Raf and C-Raf. Interestingly, the induction of signaling and cell growth both occur in a biphasic pattern, with low compound concentrations (0.01-0.1 uM) causing maximal induction, and higher compound concentrations (1-10 uM) causing less profound induction. Such a biphasic pattern is also observed in biochemical assays with purified wild-type B-Raf or C-Raf. The biphasic pattern is suggestive of a mechanism involving the interaction of two signaling subunits. In addition, recent literature data (Rajakulendran, Nature, 461:542-6) has demonstrated that Raf dimerization can upregulate pMEK, not through trans-phosphorylation of Raf molecules but presumably by conformational activation of the kinase. Consistent with that model, we show that Raf compound treatment induces B/C-Raf dimer formation in cells. In addition, knockdown of A-, B- or C-Raf with siRNA does not abrogate the Raf compound induction of pMEK and pERK, suggesting that induction might be mediated by Raf homo- as well as hetero-dimerization. Notably, knockdown of K-Ras in K-RasMUT cells also does not abolish the induction, implying that this effect is not mediated by Ras. Taken together, these data suggest a model in which compound binding to one Raf molecule induces dimerization and conformational activation of a partner Raf molecule in the dimer. These observations can explain why wild-type Raf and mutant Ras tumors are insensitive to selective Raf kinase inhibitors and might also have important implications for toxicity, since induction of strong mitogenic signaling could lead to hyperproliferation of normal tissues. Understanding the Raf compound induction mechanism may lead not only to the design of improved inhibitors, but also to methods for overcoming the induction seen with current development compounds. Toward that end, we show that combining a MEK inhibitor with a Raf compound causes inhibition of both pERK and cell growth and may therefore have significant advantages in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 20.