Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo.

Inhibitors of isoprenylcysteine carboxylmethyltransferase (Icmt) are promising anti-cancer agents, as modification by Icmt is an essential component of the protein prenylation pathway for a group of proteins that includes Ras GTPases. Cysmethynil, a prototypical indole-based inhibitor of Icmt, effectively inhibits tumor cell growth. However, the physical properties of cysmethynil, such as its low aqueous solubility, make it a poor candidate for clinical development. A novel amino-derivative of cysmethynil with superior physical properties and marked improvement in efficacy, termed compound 8.12, has recently been reported. We report here that Icmt (-/-) mouse embryonic fibroblasts (MEFs) are much more resistant to compound 8.12-induced cell death than their wild-type counterparts, providing evidence that the anti-proliferative effects of this compound are mediated through an Icmt specific mechanism. Treatment of PC3 prostate and HepG2 liver cancer cells with compound 8.12 resulted in pre-lamin A accumulation and Ras delocalization from the plasma membrane, both expected outcomes from inhibition of the Icmt-catalyzed carboxylmethylation. Treatment with compound 8.12 induced cell cycle arrest, autophagy and cell death, and abolished anchorage-independent colony formation. Consistent with its greater in vitro efficacy, compound 8.12 inhibited tumor growth with greater potency than cysmethynil in a xenograft mouse model. Further, a drug combination study identified synergistic antitumor efficacy of compound 8.12 and the epithelial growth factor receptor (EGFR)-inhibitor gefitinib, possibly through enhancement of autophagy. This study establishes compound 8.12 as a pharmacological inhibitor of Icmt that is an attractive candidate for further preclinical and clinical development.

Functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt).

The enzyme isoprenylcysteine carboxyl methyltransferase (Icmt) plays an important role in the post-translational modification of proteins involved in the regulation of cell growth and oncogenesis. The biological consequences of Icmt inhibition strongly implicate the enzyme as a potential therapeutic target for cancer and provide a compelling rationale for developing specific Icmt inhibitors as anti-cancer agents. We report here the systematic modification of the known Icmt inhibitor cysmethynil to give an analog 15 with greatly improved solubility and PAMPA permeability which was achieved with concurrent gains in Icmt inhibitory and cell-based antiproliferative activities. The modifications involved replacing the amide side chain of cysmethynil with a tertiary amine, and introducing an aminopyrimidine ring in place of m-tolyl. The presence of the weakly basic and polar aminopyrimidine ring contributed significantly to the potency and drug-like profile of the final compound.