1.       Development of “Frontier” Anti-Cancer Drugs that Overcome the Triad-of-Death: Entrance into Multi-Centre, Clinical Trials.

Richardson’s pioneering, interdisciplinary work on the design/development of metal-binding agents provided a paradigm-shifting approach to cancer treatment. Indeed, he elucidated crucial structure-activity relationships of novel ligands (chelators) through 30-years of analysis, which led to novel, patented therapeutics at the pharmacological frontier that have entered Phase-I clinical trials. In fact, these agents not only demonstrate potent and safe anti-tumour activity, but also overcome drug-resistance and metastasis, the major killers in cancer: the “triad of death” (Pharmacol.Res.2015;100:255-60).

In fact, his research and new drugs stimulated a burgeoning field that now realises the potential of targeting essential metals, which like folate, are critical nutrients for DNA synthesis. Unlike folate, which received tremendous attention as a target for anti-cancer therapy and for which a Nobel Prize was awarded, iron had been largely ignored. This was due to the lack of specifically designed ligands that demonstrated potent and selective activity and prevented whole body iron- or copper-depletion. As such, the examination of metal-binding chelators for cancer therapy was initially considered sheer folly.

However, Richardson felt differently. Despite marked resistance to such ideas, he initiated a highly integrated, interdisciplinary approach embracing: chemistry/medicinal chemistry/pharmacology/biochemistry/cell-molecular biology/animal biology/clinical trials.

To achieve breakthrough anti-cancer agents to inhibit the “triad-of-death” in cancer, namely primary tumour growth, drug-resistance and metastasis, a global approach was needed to bridge the disparate worlds of chemistry and biology and harness new knowledge, which could only be achieved by comprehensive understanding of how the agents interact at the molecular, cellular and animal levels.  Through onerous iterations of intensive structure-activity relationship studies over 30-years, starting in 1984, the ligands were designed, synthesised, biologically-tested, modified, and tested again.

In 1995, Richardson published a seminal paper (BLOOD.1995;86:4295-306;citations-323) demonstrating for the first time that aroylhydrazone ligands could be specifically designed for treating iron overload or cancer. These are two very different, but common conditions, requiring distinct drug design features. For treating iron-overload, excess iron should be removed without inducing anti-proliferative activity, while for cancer, targeting a discrete iron pool needed for DNA synthesis and causing potent anti-proliferative activity was critical.  While this could be achieved by modification of the aroylhydrazone framework, particularly via modification of lipophilicity, these ligands did not display potent enough anti-cancer activity. An extra punch was required!

1.1 Targeting the Lysosome: The “Double Punch”

Another 10-years of intensive structure-activity relationship studies were required (e.g., BLOOD.2002;100:666-676;citations:170;Clin.Cancer.Res.2003;9:402-414;citations-205) before the di-2-pyridylketone thiosemicarbazone ligands were developed, with di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) as the lead agent. These compounds displayed potent and selective activity mediated by the innovative "Double Punch" mechanism, and importantly, did not cause whole animal iron-depletion due to the low dose required (BLOOD.2004;104:1450-1458;citations-316;PNAS.2006;103:7670-5;citations-377).

The Double Punch mechanism goes well-beyond the "old-school" concept of iron/copper-binding. Indeed, these ligands were re-designed from their aroylhydrazone forerunners by replacing the hard oxygen donors with soft donors such as nitrogen and sulphur. In this way, they bind critical iron or copper (1st-Punch) that is critical for primary tumour growth. The agents bind these redox-active metals after they are liberated by the degradation of metal-containing proteins in the lysosome. They then form a cytotoxic, redox-active metal complex (2nd-Punch), which generates toxic radicals, damages the lysosome and causes lysosomal-membrane permeabilization, which kills the tumour (J.Med.Chem.2006;49:6510-6521;citations-297;Cancer.Res.2011;71:5871-80;citations-157).

1.2 Targeting the Lysosome Uncovers an Innovative Mechanism of Intracellular Resistance that can be Tricked/Hijacked and Used Against the Tumour.

Importantly, these DpT agents also overcame drug-resistance to standard chemotherapies by a mechanism that remained unknown at that time (PNAS.2006;103:7670-5), but was elucidated by Richardson through another 10-years of dogged-effort (JBC.2015;290:9588-9603;citations-52). In fact, the ability of these new agents to overcome resistance was due to their targeting of the lysosome. However, it also established a new paradigm regarding the mechanism how drug-efflux pumps, such as P-glycoprotein (Pgp), actually functon to impart resistance.

Notably, P-gp was generally thought to be only on the plasma membrane, where it transports cytotoxic drugs like doxorubicin (DOX) out of the cell, preventing their damage to their major target, nuclear DNA (Fig. 1). However, this model does not take into account that the plasma membrane becomes endocytosed, leading to endosomes and then lysosomes (JBC.2013:288:31761-7;citations-77).

Richardson’s paradigm-shifting concept was that not only are drug-pumps active on the cell surface, but they are ALSO active in lysosomes (Fig. 1). Once endocytosed into these organelles, the pumps no longer face out, but into the lumen (Fig. 1). Indeed, he demonstrated that these pumps act to transport cytotoxic drugs into lysosomes, where they sequester DOX away from its sensitive targets (e.g., nuclear DNA), resulting in resistance to DOX. This led to the novel concept of lysosome-mediated intracellular resistance (JBC.2013:288:31761-71; Fig. 1).

These findings provided the foundation for understanding how Dp44mT overcame resistance.  In fact, Richardson demonstrated his drugs were Pgp substrates and were not only pumped out of the cell by Pgp at the plasma membrane, but also transported by Pgp into lysosomes (JBC.2015;290:9588-9603; Fig.2).

This facilitated access to their major target, the lysosome. Once in the lysosome, Dp44mT binds metals liberated by degradation of iron or copper complexes, forming redox-active metal complexes. These then generate reactive oxygen species facilitating lysosomal-membrane permeabilization leading to cell death (Fig.2). Thus, this overcomes Pgp-resistance in-vitro and in-vivo (JBC.2015;290:9588-9603).  

Hence, the endogenous machinery of the tumour cell (Pgp) that is used to protect itself against standard chemotherapies, could be “tricked/hijacked” by Richardson’s drugs and used AGAINST the cancer to overcome resistance. This is a critical property, as drug-resistance alone resulted in 7.6-million deaths in 2008 and this is expected to increase to 13.1-million in 2030 (http://www.wpro.who.int/mediacentre/factsheets/fs_20120926e/en/).

1.3 Further Endogenous Biochemical Machinery Harnessed Against Tumours to Block Metastasis

Additionally, through gene array analysis, Richardson discovered that through their ability to bind cellular iron, these novel drugs induce expression of an exciting metastasis suppressor protein, called N-myc downstream regulated gene-1 (NDRG1) in tumours, but not normal tissue (BLOOD.2004;104:2967-75;citations-240; PNAS.2006;103:7670-5). This up-regulation of NDRG1 occurs through cellular iron-depletion activating the transcription factor, hypoxia-inducible factor-1α (HIF-1α), which transcribes NDRG1 (BLOOD.2004;104:2967-75). At that time, the mechanism how NDRG1 inhibits metastasis was unknown.

Considering this, through and additional 15 years of research, Richardson elucidated that NDRG1 acts as a potent anti-oncogenic signaling inhibitor, by blocking multiple key metastatic/oncogenic pathways e.g., AKT/PI3K, TGFβ, NFĸB, RAS/MAPK, FAK/paxillin, WNT, ROCK/pMLC2,etc; Mol. Pharmacol.2012;83:454-69;Antioxid.Redox.Signal.2013;18:874-87;Br.J.Cancer.2013;108:409-19;J.Cell.Sci.2014;127:3116-30;Mol.Pharmacol.2016;89:521-40;Mol. Pharmacol.2017;91:499-517), up-regulating the tumor suppressor, PTEN (Antioxidants.Redox.Signaling.2013;18:874-87;citations-85), and down-regulating the cyclin-dependent-kinase inhibitor, p21 (Carcinogenesis.2011;32:732-740;citations-83). Furthermore, NDRG1 inhibited the crucial step in metastasis, the epithelial-mesenchymal transition (JBC.2012;287:17016-28;citations-131). 

 How could NDRG1 elicit such broad anti-oncogenic activity over so many signalling pathways? Richardson demonstrated this occurred through inhibition of key upstream signalling proteins that are the “Kings and Queens” of oncogenic signalling, namely EGFR, HER2 and HER3. By increasing their degradation, NDRG1 acts as an anti-oncogenic “blanket” to suppress multiple, potent, downstream signaling pathways (JBC.2016;291:1029-52;citations-22).

Despite its breakthrough properties of inhibiting primary tumour growth, metastasis and resistance, unfortunately, at non-optimal doses, Dp44mT led to cardiotoxicity (PNAS.2006;103:7670-5). Thus, additional work was required to overcome this.

A further 5-years of structure-activity analysis led to DpC, which could be administered at far higher doses and did not induce cardiotoxicity (Mol.Pharmacol.2011;80:598-609;citations-107). DpC was also active intravenously and orally, which is critical for convenient dosing (J.Med.Chem.2012;55:7230-44;citations–98). Significantly, DpC was far more active against aggressive pancreatic cancer, than the “gold-standard” therapy, gemcitabine, or Dp44mT, and showed synergism with standard chemotherapy (J.Med.Chem.2012;55:7230-44). 

Hence, frontier patented agents inhibiting the triad-of-death were developed with properties not matched by established chemotherapies. Richardson then single-handedly, commercialised DpC as Executive Director of Oncochel Pty-Ltd. DpC was licensed to CTHULHU VENTURES USA, leading to Oncochel Therapeutics LLC/Oncochel Therapeutics Pty-Ltd that were established to take DpC into trials. The long-term impact is that DpC has gone through extensive evaluation in multiple species as a new anti-cancer drug and has entered multi-centre, clinical trials.

The development of DpC demonstrated chelators can be designed for cancer and this stimulated a new branch of pharmacology realising iron as a key target. Indeed, the potent anti-tumour/anti-metastatic activity of DpC and Dp44mT have been repeatedly verified by others (Cancer.Res.2009;69:948-57;EMBO.Mol.Med.2012;4:93-108;Am.J.Transl.Res.2016;8:5370-85; Oncol.Lett.2018;15:7999-8004; etc.).

This advance is clinically and pharmacologically paradigm-shifting, as it is a frontier therapeutic strategy. No other anti-cancer drug possesses this mechanism, nor can it potently overcome Pgp-mediated-resistance and inhibit metastasis, which are THE major killers in cancer.

This all-embracing strategy to develop anti-cancer agents, also led to exciting paradigm-shifting discoveries on other molecules and diseases related to metal metabolism. For example:

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(1) investigating mechanisms of iron uptake by cancer cells - from cells to transgenic mice (JBC.1992;267:13972-9;EMBO.J.1995;14:4178-86;BLOOD.2006;107:2599-2601) and elucidation why they are so sensitive to iron-depletion (BLOOD.1997;89:3025-38;BLOOD.1999;94:781-92);

(2) dissecting mechanisms of mitochondrial iron-overload and its role in cardio-degeneration (PNAS.2008;105:9757-9762;PNAS.2009;106:16381-16386;PNAS.2012;109:20590-5); and

(3) demonstration the critical diatomic signalling gas, nitric oxide, is not freely diffusible in cells, but rigorously controlled via transportation and storage as a dinitrosyl-dithiol-iron complex by MRP1 and GSTP1 (PNAS.2006;103:7670-7675;JBC.2012;287:6960–6968;JBC.2016;291:27042–61).

In conclusion, these studies constitute an intimate contribution by Richardson (93% of his 4-publications are as first/senior/corresponding author) and his team, which included >170-fellows/RAs/students. Due to his mentorship, these trainees have become outstanding scientists themselves.