Preclinical programs


  • HER2+ Breast Cancer

  • Triple Negative Breast Cancer

  • Other Cancers

Rare Diseases

  • Wilson's Disease

  • Rett Syndrome

PTP1B Enzyme / HER2+ Breast Cancer

Disruption of the normal patterns of protein phosphorylation results in aberrant regulation of signal transduction and has been implicated in the etiology of a variety of major human diseases. The ability to modulate such signaling pathways selectively holds enormous therapeutic potential. Currently, the focus of effort in this aspect of drug development has been on protein kinases, particularly protein tyrosine kinases (PTKs); however, this ignores the other major component of phosphorylation-dependent regulation of signaling. Protein phosphorylation is a reversible process, in which the coordinated and competing activities of kinases and phosphatases are important for determining signaling outcome. Nevertheless, the protein tyrosine phosphatases (PTPs) remain a largely untapped resource for drug development. Since its discovery 25 years ago, PTP1B has become a highly validated therapeutic target. A major breakthrough was the definition of the role of PTP1B in down-regulating insulin and leptin signaling. In mice, targeted deletion of the PTPN1 gene, which encodes PTP1B, produced healthy animals that displayed increased insulin sensitivity and resistance to obesity induced by a high fat diet. Thus, inhibitors of PTP1B may promote signaling in insulin- and leptin-resistant states and offer a novel approach to treating diabetes and obesity. Nevertheless, the function of PTP1B is not restricted to metabolic regulation; it is over-expressed in breast tumors together with the PTK HER2. Mice expressing activated alleles of HER2 in mammary glands develop multiple mammary tumors and frequent metastases to the lung; however, when such mice were crossed with PTP1B-null mice, tumor development was delayed and the incidence of lung metastases was decreased. Conversely, targeted overexpression of PTP1B alone was sufficient to drive mammary tumorigenesis. These observations suggest that, in addition to its role as a negative regulator of insulin and leptin signaling, PTP1B plays a positive role in promoting signaling events associated with breast tumorigenesis. Therefore, inhibition of PTP1B function may represent a novel therapeutic strategy not only to address diabetes and obesity, but also mammary tumorigenesis and malignancy.

In light of these breakthroughs, there have been major programs in industry focused on developing small molecule inhibitors of PTP1B. These efforts, which followed standard procedures of targeting the active site of PTP1B, have been frustrated by technical challenges arising from the chemistry of PTP catalysis. Although it was possible to generate potent, specific and reversible inhibitors of PTP1B, such molecules were highly charged and thus of limited drug development potential. In contrast, DepYmed is overcoming these challenges by focusing on allosteric inhibitors that bind at unique sites remote from the catalytic center of the enzyme. PTP1B was purified originally from human placenta as a 37kDa catalytic domain, which has been the focus of attention to date for mechanistic analysis, as well as for drug screening. Nevertheless, PTP1B exists in vivo as a longer protein of ~50kDa, in which the C-terminal segment, which is deleted from the 37kDa protein, serves a regulatory function. We have demonstrated that an aminosterol natural product, MSI-1436/Trodusquemine, inhibited the full-length form of PTP1B preferentially in a reversible, selective manner. We have identified the binding sites for MSI-1436 in PTP1B and defined the mechanism of inhibition. By targeting the unique, C-terminal, non-catalytic segment that is unrelated to any other member of the PTP family, such allosteric inhibitors would have the potential to be highly specific for PTP1B. Furthermore, we have demonstrated that by targeting PTP1B, MSI-1436 attenuated HER2 signaling, resulting in extensive inhibition of tumor growth and abrogation of metastasis to the lung in HER2-positive animal models of breast cancer. Overall, these data establish that PTP1B is a bona fide target for therapeutic intervention in HER2-positive cancer and illustrate a novel mechanism for specific inhibition of PTP1B through which such intervention may be achieved.

Although the first drugs directed against protein tyrosine kinases (PTKs) represent breakthroughs in cancer therapy, challenges remain. For example, the humanized antibody Herceptin (Trastuzumab) targets the PTK HER2, which is amplified and/or overexpressed in ~25% of breast tumors, where it associated with poor prognosis. Herceptin is the treatment of choice for HER2-dependent cancer, although there are problems with de novo and acquired resistance. Similar problems have limited the success of other PTK-directed inhibitors. Therefore, it is anticipated that alternative therapies, to target simultaneously different signaling enzymes and processes, may be more effective than targeting individual PTKs alone. Currently, we are testing whether inclusion of MSI-1436 together with Herceptin, to target simultaneously the PTK and its downstream signaling, will prolong the time to development of resistance, or even overcome resistant states.

Breast cancer, which is the most prevalent malignancy in women, is a heterogeneous disease that can be categorized into different subtypes based on expression of particular markers. They are luminal A and B (which are hormone receptor-positive (estrogen and progesterone receptors)), HER2-positive (which are defined by high levels of the HER2 protein tyrosine kinase and are hormone receptor-negative), triple-negative (which is a heterogeneous group that is both HER2- and hormone receptor-negative, and shows similarities to cancers that are classified as basal-like) and normal-like (which is similar to luminal cancer being hormone receptor-positive, but HER2-negative). Currently, prognosis and therapeutic strategies vary for each subtype.

A variety of drugs that target HER2 have been developed, including antibodies such as Herceptin/Trastuzumab, antibody conjugates such as T-DM1/Kadcyla and small molecule drugs such as Lapatinib. Although these target the cancer cell with specificity, there remain problems of resistance, both intrinsic and acquired, that limit the effectiveness of such therapies. At this time, inhibitors of PTP1B show potential as a novel approach to treatment of HER2-positive cancer, either alone or in combination with Herceptin.

In contrast, triple-negative breast cancer, which comprises ~15% of all breast cancer, is aggressive and characterized by poor prognosis; it is treated with combinations of surgery, radiation and chemotherapies. It has been reported that there are distinct tyrosine phosphorylation-dependent signatures associated with basal breast cancer cells, in particular featuring the SRC family of PTKs. Nevertheless, these remain to be exploited for targeted therapy and further insights into the signaling changes associated with the various breast cancer subtypes are being explored. Interestingly, recent studies have suggested that elevated levels of copper may underlie signaling changes in various cancers.

Copper is an essential element in living organisms, the functional significance of which is enhanced because of its ability to adopt either reduced (Cu+) or oxidized (Cu2+) states. There are a wide variety of copper-dependent enzymes, several of which have been implicated different aspects of cancer. Examples include cytochrome c oxidase, the terminal protein in the mitochondrial electron transport chain that plays a critical role in ATP synthesis, and lysyl oxidase, which cross-links the extracellular matrix and may be important in metastasis, More recently, it has been implicated in the regulation of signal transduction through control of the activity of kinases such as MEK, linking copper to the control of cell growth, and its disruption in tumorigenesis and metastasis. Elevated levels of copper have also been noted in sera of a variety of cancer patients; this has led to the testing of copper chelators, including tetrathiomolybdate, in various tumor models and in clinical trials, with encouraging results.

Wilson's Disease


Metals play critical roles in the control of protein function that introduce novel aspects of chemistry and structure in biological systems. Copper is an essential element in living organisms, the functional significance of which is enhanced because of its ability to adopt either reduced (Cu+) or oxidized (Cu2+) states. There are a wide variety of copper-dependent enzymes. Recently, copper has been implicated in the regulation of signal transduction through control of the activity of kinases, such as MEK, linking copper to the control of cell growth and its disruption in tumorigenesis and metastasis. Although fundamentally important to normal cell function, copper can also be toxic, indicating that its levels must be tightly regulated, both in the organism as a whole and at the cellular level.

In mammals, diet serves as the primary source of copper. It is absorbed in the gut and transported to the liver bound to albumin, then distributed to other tissues through the bloodstream in a complex with ceruloplasmin. The levels of copper are under complex homeostatic control, including transporters that control influx and efflux, together with specialized chaperones that deliver the metal to its sites of action. Within the cell, the levels of free copper are kept to a minimum by chaperones that direct the metal to particular targets. This includes Cu-ATPases such as ATP7B, which serves a dual function, either delivering copper to the secretory pathway where it can be incorporated into copper-dependent enzymes, or under conditions in which copper is present in excess, exporting it from the cell. Disruption of these homeostatic mechanisms is associated with a variety of disease states. In particular, mutations in ATP7B lead to accumulation of the metal resulting in Wilson’s disease.

The incidence of Wilson’s disease is estimated to be 1 in 30,000 worldwide, although this may be an underestimate as its symptoms overlap with other conditions. Wilson’s disease is a severe autosomal recessive disorder and, to date, more than 300 disease-causing mutations have been identified in the ATP7B gene. The physical burden of the disease is felt in the liver, in particular, as this tissue expresses high levels of ATP7B. It begins with a presymptomatic period, during which copper accumulates in the liver. If diagnosis occurs at this stage, the prognosis is good with current therapies; however, without treatment, a variety of hepatic problems are encountered from enlargement of the liver, to hepatitis and cirrhosis, and even acute liver failure. As the disease progresses further, it results in the development of speech and cognitive impairment, particularly tremors and dystonia, as well as ataxia and Parkinsonism. In addition, psychiatric problems including personality changes, antisocial behavior, anxiety and depression appear in Wilson’s patients at some time during the course of the disease. Most patients with neurological symptoms also develop Kayser-Fleischer rings, which are formed by copper deposits in the cornea, leading to a brown discoloration that can be diagnostic for the disease.

If Wilson’s disease is diagnosed in the early, pre-symptomatic phase, the prognosis is good with current therapeutics. Nevertheless, early diagnosis is challenging and treatment becomes more difficult as the course of the disease progresses; ultimately, patients may develop fulminant hepatic failure that requires liver transplantation for resolution. The primary therapeutic strategy is to try and reduce the levels of copper in the patient and re-establish homeostasis. There are two approaches to reducing copper levels. Zinc salts, such as zinc acetate, are a first line of therapy, particularly for asymptomatic patients, and are also important as a maintenance therapy for long-term management of Wilson’s disease after treatment with chelators. Zinc decreases absorption of copper from the gut by inducing expression of metallothionein in intestinal cells, which then traps both metals leading to excretion as the mucosal cells are sloughed off and removed in the feces. Although this is less toxic than the existing chelators, it does not have the same ability to remove copper from tissues that have become overloaded. Consequently, chelating agents that bind copper directly in the tissues or blood and facilitate its excretion have been developed for use as an initial ‘de-coppering” step. Currently, the standard of care for Wilson’s disease includes treatment with the chelating agents D-penicillamine or trientine, with tetrathiomolybdate progressing as an experimental therapy in clinical trials.

D-penicillamine, also known as Cuprimine, promotes urinary excretion of copper; however, is not specific for copper and is a general chelator that has been used to treat heavy metal poisoning. Neurological problems associated with D-penicillamine are a particular concern and it has been reported that the deterioration encountered may be irreversible. Other problems include swelling and inflammation of kidneys, hepatotoxicity and hematological abnormalities, conditions that may be exacerbated by chronic administration regimens. In light of the problems with D-penicillamine, trientine has been used more extensively. Trientine, also known as Syprine, has a polyamine-like structure that is distinct from D-penicillamine. Although it forms a complex with copper, it has been reported also to bind iron and zinc in vivo. It facilitates copper excretion in the urine and is also suggested to impair intestinal absorption and increase excretion in feces.  A problem with trientine is that it has a short half-life in humans. It is poorly absorbed from the GI tract and what is absorbed is rapidly metabolized, via acetylation, and inactivated. Although its side effects are less marked than those of D-penicillamine, toxicity problems have been reported. More recently, tetrathiomolybdate has progressed in clinical trials. It forms a complex with copper that restricts absorption from the gut and limits cellular uptake, eliminating copper in urine and feces. When used initially as an ammonium salt, problems were encountered with stability of the compound. Subsequently, a bis-choline salt of tetrathiomolybdate, also known as Decuprate/WTX101, has been tested in phase 2 clinical trials. Although results were encouraging, some serious adverse events were noted and further trials will be necessary to establish whether this will be an effective therapeutic agent.

Clearly, new approaches are required to treat Wilson’s disease. We have identified a portfolio of small molecules that have several advantageous properties, including that they are highly specific for copper, are orally bioavailable, crosses the blood-brain barrier and promotes fecal excretion of the metal. The compounds in the portfolio inhibited copper-induced cell death in cell models of Wilson’s disease, including fibroblasts derived from patients in which elevated copper had been demonstrated. Furthermore, when the compounds are administered orally or intraperitoneally to the toxic milk mouse model of Wilson’s disease, they lowered copper levels in the liver and brain. Our data suggest the new small molecules may offer advantages as a therapeutic agents to deplete the excess metal in Wilson’s disease. In addition, it is now becoming evident that abnormally high levels of copper may have an impact in other indications including cancer and neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease, suggesting further potential applications for a number of compounds in DepYmed's portfolio.

Rett syndrome (RTT) is an X-linked neurological disorder that affects ~1 in 10,000 female births, a similar incidence to cystic fibrosis and Huntington’s disease. Girls with Rett syndrome develop normally during the first six months of life, but then begin to present a host of neurodevelopmental abnormalities including learning disabilities, loss of motor skills, stereotypic hand movements, irregular breathing and seizures. Boys are more severely affected and tend not to survive infancy due to encephalopathy. Rett syndrome patients also exhibit deficits in social interactions, a feature reminiscent of autism.

In >95% of cases, Rett syndrome is caused by loss-of-function mutations in the X-linked gene MECP2, which was the first autism spectrum disorder gene to be identified. MECP2 regulates chromatin structure and the expression of a wide range of genes throughout the genome, which has focused attention on its function as a regulator of gene expression. The development of mice lacking an intact Mecp2 gene was a breakthrough for the field. These mice have been shown to recapitulate a broad spectrum of phenotypes, with similarities to those encountered in Rett syndrome patients. As in human patients, deficits are more pronounced in males than females. Most importantly, there have been studies to show that after disease onset in mice lacking the gene, restoration of MECP2 expression can rescue most neurological deficits and improve survival. This indicates that symptoms associated with Mecp2 loss can be reversed, suggesting that this may be a treatable disease.

These broad effects represent a challenge for drug development and currently there is no disease modifying therapy to correct dysfunctional MECP2 in Rett syndrome. Instead, the focus has been on managing symptoms and identifying druggable targets that function downstream of MECP2, including signaling events that may be subject to its influence. The neurotrophic factor BDNF (Brain-Derived Neurotrophic Factor) was identified as a target of MECP2, the levels of which are decreased in Rett syndrome. Experimental approaches to elevate BDNF did ameliorate some symptoms and alternative approaches utilizing BDNF-mimetics are being tested. Levels of Insulin-like Growth factor-1 (IGF-1) are also reduced in Mecp2-mutant mice and treatment with an N-terminal tripeptide derived from IGF-1 has been reported to ameliorate some symptoms of Rett syndrome in these animals, leading to clinical trials.


We noted that obesity and leptin-resistance has been reported in some mouse models and insulin resistance has been reported in some Rett patients. Interestingly, obesity is becoming recognized as a common feature of autism spectrum disorders. We observed that glucose metabolism and insulin signaling in the brain were attenuated in Mecp2-mutant mice; this suggested to us the possibility of a role for the protein tyrosine phosphatase PTP1B, which is recognized as a major metabolic regulator that is known to attenuate both insulin and leptin signaling. PTP1B negatively regulates the PI3K/AKT signaling pathway that is important for development and is attenuated in Rett syndrome models.  We demonstrated that the PTPN1 gene, which encodes PTP1B, was a direct target of MECP2 and that the levels of PTP1B protein were dramatically increased in Mecp2-mutant mice and in fibroblasts derived from Rett syndrome patients. Furthermore, we showed that administration of small molecule inhibitors of PTP1B to Mecp2-mutant mice dramatically extended the lifespan of Mecp2-/y male mice and improved performance of Mecp2-/+ female mice in behavioral assays. Our data illustrate that PTP1B recognized TRKB as a direct substrate and that inhibition of PTP1B resulted in enhanced TRKB phosphorylation in the brain of Mecp2-mutant mice. This validates PTP1B as a mechanism-based target for therapeutic intervention in Rett syndrome – the elevated PTP1B in Rett syndrome may represent a barrier to normal BDNF/TRKB function. This suggests a new strategy for treating this disease by modifying signal transduction pathways with small molecule drugs that target PTP1B.