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Cancer treatment has been considerably advanced by the relatively recent development of small molecules capable of inhibiting oncogenic kinase signaling, but exogenous enhancement of tumor suppressive signaling remains elusive. Here, we sought to design small molecules capable of activating the tumor suppressor kinase LKB1. Designing compounds capable of increasing kinase activity has been more structurally challenging than those aimed at inhibiting them: apart from the ATP-binding pocket, there is rarely a site that would be conducive to binding a small molecule. Unlike most protein kinases, LKB1 signals as part of an obligate trimer consisting of itself, a scaffolding protein (MO25), and a pseudokinase (STRAD). As part of its mechanism of activation, LKB1 must bind to an ATP-bound STRAD to take on its active kinase conformation. Targeting STRAD provides a unique opportunity to allosterically stabilize and enhance LKB1 kinase activity. Using a structure-based drug design, we developed compounds that are able to selectively bind STRAD's ATP-binding pocket. We confirmed that our STRAD targeting small molecules are able to enhance the activity of recombinant LKB1 in a kinase assay. The observed increase in LKB1's kinase activity corresponds to increased association of complex components after drug treatment, as suggested by immunoprecipitation of the trimer. To understand the clinical contexts in which our compounds might be most effective, we performed a viability screen across multiple histologies to determine which cancer cells could be sensitive to LKB1 activation. Sensitive cells were used to investigate the mechanism of action of exogenous LKB1 stimulation. LKB1 signals through members of the AMPK-related kinase family to carry out its tumor suppressive functions in the cell. Thus, we used western blot analysis of LKB1 proximal and distal mediators to assess changes in downstream signaling. We found that activation of multiple LKB1 effectors was dose dependent and occurred rapidly, with signal enhancement seen in more tumor-relevant low adherence cell culture conditions. Real-time microscopy confirmed that our compounds slowed cell proliferation in a dose-dependent manner. This work demonstrates that targeting a pseudokinase with a small molecule to allosterically activate of a tumor suppressor kinase is possible, therapeutically effective in vitro, and triggers multiple downstream signaling pathways to decrease cancer cell proliferation. Citation Format: Dominique C. Mitchell, Jin Liu, Zheng Wang, Changliang He, Luo Ding, Marc Adler, LeeAnn L. Wang, Aidan Keith, Y. Christina Hwang, Tsz Kin M. Tsui, Roopa Ramamoorthi, Sarah Lively, Robert A. Drakas, Vijay Ramani, Kliment A. Verba, Tingting Qing, Richard T. Beresis, John D. Gordan. Small molecule activation of the LKB1 tumor suppressor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5228.

Graphical abstract, Highlights • WIPO Re:Search leverages pharmaceutical assets to advance anthelmintic research. • BIO Ventures for Global Health (BVGH) facilitates collaborations and capacity building. • Novartis scientists trained a Cameroonian researcher on advanced biochemistry skills. • Researchers from Canada and Cameroon partnered to discover onchocerciasis drugs. • Natural products were screened against schistosomes and soil-transmitted helminths., Neglected tropical diseases (NTDs), malaria, and tuberculosis have a devastating effect on an estimated 1.6 billion people worldwide. The World Intellectual Property Organization (WIPO) Re:Search consortium accelerates the development of new drugs, vaccines, and diagnostics for these diseases by connecting the assets and resources of pharmaceutical companies, such as compound libraries and expertise, to academic or nonprofit researchers with novel product discovery or development ideas. As the WIPO Re:Search Partnership Hub Administrator, BIO Ventures for Global Health (BVGH) fields requests from researchers, identifies Member organizations able to fulfill these requests, and helps forge mutually beneficial collaborations. Since its inception in October 2011, WIPO Re:Search membership has expanded to more than 90 institutions, including leading pharmaceutical companies, universities, nonprofit research institutions, and product development partnerships from around the world. To date, WIPO Re:Search has facilitated over 70 research agreements between Consortium Members, including 11 collaborations focused on anthelmintic drug discovery.

Schistosomiasis, one of 17 diseases deemed to be neglected by the World Health Organization, has received little attention from the biopharmaceutical industry. Due to this, only a handful of drugs have been developed to treat schistosomiasis, with only one, praziquantel, used in most endemic regions. Growing concern over resistance coupled with praziquantel's incomplete efficacy across all stages of the Schistosoma platyhelminth life cycle highlights the urgent need for new drugs. The WIPO Re:Search consortium is a platform whereupon biopharmaceutical company compounds are being repurposed to efficiently and cost-effectively develop new drugs for neglected diseases such as schistosomiasis. This article summarizes recent clinical-stage efforts to identify new antischistosomals and highlights biopharmaceutical company compounds with potential for repurposing to treat schistosomiasis.

1BIO Ventures for Global Health, Seattle, WA, USA 2WIPO, Geneva, Switzerland *Author for correspondence: Tel.: +1 206 732 2131 E-mail: jdent@bvgh.org Advances in science, medicine and technology have enabled high-income countries to dramatically reduce the burden of infectious diseases and even eliminate some of them from their populations. Developing countries, however, are still struggling with high rates of preventable morbidity and mortality due to infectious diseases. Neglected tropical diseases (NTDs), malaria and tuberculosis have a devastating impact on more than 1.4 billion people living in poverty (Table 1) [1], yet current treatments are often expensive, outdated, ineffective and burdened with serious side effects. More effective drugs, vaccines and simple point-of-care diagnostics are needed. Success in developing these new products requires product development know-how, and access to compound libraries and state-of-the-art reagents, as well as an understanding of regulatory processes. Innovative research and product development in the pharmaceutical sector is extremely expensive. The cost of bringing a new drug to market is often in excess of US$1 billion [101]. Recovering these costs through active IP protection has always been central to the pharmaceutical industry’s business model, making it possible to satisfy and return value to shareholders. Protection of IP is thus actively pursued in the pharmaceutical and biotechnology sectors to ensure a period of exclusivity for products brought to market. IP, including patents and trade secrets, guide a company’s strategic direction and make it possible to establish a competitive advantage. These assets are shared or acquired through strict licensing agreements and often at great financial expense to the licensee commensurate with the perceived commercial potential. Gaining access to the assets of pharmaceutical and biotechnology companies has been quite a challenge for researchers working in noncommercial contexts.

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicum pc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified.

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which blocks matrix metalloproteinase (MMP) activity. TIMP-1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP-1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl4), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti–TIMP-1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl4-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle α-actin (α-SMA). Compared to controls, rats administered anti–TIMP-1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti–TIMP-1 resulted in a marked decrease in α-SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion, administration of a TIMP-1 antibody attenuated CCl4-induced liver fibrosis and decreased HSC activation and MMP-2 activity. (HEPATOLOGY 2004.)

Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ). The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M. extorquens AM1 PqqF has low identity with the same endopeptidase family. M. extorquens AM1 pqqE complemented a K. pneumoniae pqqF mutant.

Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino-acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly.

Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes). Subcloning and transposon mutagenesis experiments have assigned these mutants to five complementation groups in two gene clusters. Representatives of each complementation group were shown to lack detectable PQQ in the growth medium and in cell extracts and to contain methanol dehydrogenase polypeptides that were inactive. Therefore, these mutants all appear to be defective in PQQ biosynthesis. PQQ biosynthesis mutants of Methylobacterium organophilum DSM 760 and M. organophilum XX were complemented by using M. extorquens AM1 subclones, and PQQ biosynthesis mutants of M. extorquens AM1 and M. organophilum XX were complemented by using M. organophilum DSM 760 subclones. This analysis suggested that a total of six PQQ biosynthesis complementation groups were present in M. extorquens AM1 and M. organophilum DSM 760. A 2-kb M. extorquens AM1 DNA fragment that complemented the MoxO class of PQQ biosynthesis mutants was sequenced and found to contain two complete open reading frames and the N-terminal sequence of a third. These genes designated pqqDGC, had predicted gene products with substantial similarity to the gene products of corresponding pqq genes in Acinetobacter calcoaceticus and Klebsiella pneumoniae. pqqD encodes a 29-amino-acid peptide which contains a tyrosine residue and glutamate residue that are conserved in the equivalent peptides of K. pneumoniae, PqqA (23 amino acids), and A. calcoaceticus, PqqIV (24 amino acids), and are thought to be the precursors for PQQ biosynthesis. The organizations of a cluster of five PQQ biosynthetic genes appear to be similiar in four different bacteria (M. extorquens AM1, M. organophilum DSM 760, K. pneumoniae, and A. calcoaceticus). Our results show that a total of seven pqq genes are present in M. extorquens AM1, and these have been designated pqqDGCBA and pqqEF.