Donald William Ludovici

TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 μM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of α1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

Evolution of anti-HIV drug candidates. Part 3: diarylpyrimidine (DAPY) analogues

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.

Evolution of anti-HIV drug candidates. Part 2: Diaryltriazine (DATA) analogues.

A synthesis program directed toward improving the stability of imidoyl thiourea based non-nucleoside reverse transcriptase inhibitors (NNRTIs) led to the discovery of diaryltriazines (DATAs), a new class of potent NNRTIs. The synthesis and anti-HIV structure-activity relationship (SAR) studies of a series of DATA derivatives are described.

Evolution of anti-HIV drug candidates. Part 1: From α-Anilinophenylacetamide (α-APA) to imidoyl thiourea (ITU)

Abstract Stemming from work on a previous clinical candidate, loviride, and other α-APA derivatives, a new series of potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been synthesized. The ITU analogues, which contain a unique diarylated imidoyl thiourea, are very active in inhibiting both wild-type and clinically important mutant strains of HIV-1.

On the detection of multiple-binding modes of ligands to proteins, from biological, structural, and modeling data

There are several indications that a given compound or a set of related compounds can bind in different modes to a specific binding site of a protein. This is especially evident from X-ray crystallographic structures of ligand-protein complexes. The availability of multiple binding modes of a ligand in a binding site may present an advantage in drug design when simultaneously optimizing several criteria. In the case of the design of anti-HIV compounds we observed that the more active compounds that are also resilient against mutation of the non-nucleoside binding site of HIV1-reverse transcriptase make use of more binding modes than the less active and resilient compounds.

Abstract 4747: Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer

Fatty Acid Synthase (FASN) is a multi-domain protein that carries out de novo fatty acid (palmitate) synthesis from acetate and malonate in mammalian cells. FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. FASN has been demonstrated to play an important role in carcinogenesis by protecting cells from apoptosis. Herein we report a new series of potent, selective and orally bioavailable FASN inhibitors. Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that a spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of losartan, an older approved agent from the same drug class. This hypothesis led to a new spirocyclic imidazolinone based FASN inhibitors. Extensive SAR efforts resulted in FASN inhibitors with potent enzyme and cell activity, selectivity, and oral bioavailability exemplified by JNJ-54302833. JNJ-54302833 is a potent inhibitor of human FASN (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced medium. This cellular activity can be rescued by addition of palmitate, demonstrating on-target effects. JNJ-54302833 is also potent in many other cells, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposures. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with JNJ-54302833 resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This novel series potently inhibits the FASN KR domain (IC50 = 54 nM for JNJ-54302833); specific binding to KR was confirmed by crystal structures.In summary, we have designed and discovered a new series of FASN inhibitors that are potent both in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Additionally, palmitate rescue of lipid-reduced cellular activity suggests selectivity and pharmacodynamics studies confirm target engagement. Citation Format: Tianbao Lu, Richard Alexander, Gilles Bignan, James Bischoff, Peter Connolly, Max Cummings, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer. [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 4747. doi:10.1158/1538-7445.AM2014-4747

Abstract 801: Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment

Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively ( In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [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 801. doi:10.1158/1538-7445.AM2014-801

Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

Abstract 2535: Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors

De novo synthesis of fatty acids in mammalian cells is catalyzed by Fatty Acid Synthase (FASN), a complex dimeric protein with seven catalytic domains that processes acetyl-CoA and malonyl-CoA into palmitic acid. Palmitate and other fatty acids are essential for normal physiological processes like energy storage and production and are key intermediates in the biosynthesis of hormones and other essential biomolecules. The role of FASN in cancer has been well documented over the past decade. While FASN is expressed at low levels in most normal tissue (except liver and adipose tissue), it is highly expressed in many tumors including prostate, breast, and colon. FASN overexpression and activity is correlated with poor prognosis and disease progression. Numerous publications describing anti-cancer effects of small molecule FASN inhibitors have appeared in the scientific and patent literature. Recently, several groups reported anticancer and antiviral activities of N-benzoyl arylpiperidine FASN inhibitors. In this work we describe the discovery of novel N-benzoyl arylpiperidine and arylazetidine compounds that potently inhibit FASN enzymatic activity in vitro and antiproliferative activity in FASN-sensitive cell lines. We conducted focused structure-activity (SAR) studies exploring the benzoyl group and the aryl substituent on the piperidine or azetidine heterocycle to optimize potency, improve properties and oral bioavailability, and achieve in vivo FASN-driven pharmacodynamic (PD) activity. X-ray crystal structures of these molecules confirm a unique binding mode within the KR subdomain of FASN. Using structural information and molecular modeling, we were able to rationalize SAR trends and design FASN inhibitors with excellent in vitro potency and cellular activity. These efforts resulted in the discovery of compounds exemplified by JNJ-54380482, a potent FASN inhibitor (IC50 = 26 nM) that inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (LRM, IC50 = 8.9 nM). Engagement of FASN in cellular assays is confirmed by rescue of proliferation upon addition of palmitate. JNJ-54380482 is orally bioavailable in mice (F% = 51) and exhibits excellent plasma exposure when dosed at 10 mg/kg (Cmax = 2.6 µM, T1/2 = 5 h). In a PD study using H460 lung xenograft-bearing mice, oral treatment with 30 mg/kg of JNJ-54380482 results in 36-fold higher tumor levels of malonyl-CoA vs vehicle. In summary, we synthesized several hundred compounds having the N-benzoyl arylpiperidine and arylazetidine chemotypes. Utilizing structural information from X-ray co-crystals to understand SAR and guide the design of more potent molecules, we identified >100 analogs with highly potent FASN enzymatic (IC50 ≤ 50 nM) and antiproliferative activities in A2780 cells in LRM (IC50 ≤ 100 nM). Key examples from these series have good in vitro properties, are orally bioavailable, and demonstrate FASN target engagement in a mouse PD model. Citation Format: Peter J. Connolly, Gilles Bignan, James Bischoff, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Tianbao Lu, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Structure-activity relationships of novel N-benzoyl arylpiperidine and arylazetidine FASN inhibitors. [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 2535. doi:10.1158/1538-7445.AM2014-2535