Lysyl oxidase (LOX) is an enzyme that catalyzes the conversion of lysine to allysine, a required material for collagen cross-linking in extracellular matrix formation.  There are four LOX paralogs, denoted as lysyl oxidase like (LOXL) enzymes 1 through 4.  Both LOX and its paralogs are implicated in a wide range of diseases.

The basic structure of the LOX proenzyme is shown in the image below.  As expressed into the extracellular space, it consists of the catalytic domain to the right of the cleavage point.  The catalytic domain contain a lysyl tyrosine quinone region (denoted LTQ) and a copper binding region.  Ionized copper is a cofactor and the enzyme cannot be activated absent the presence of copper.  Cleavage is caused by the same PCP enzyme responsible for cleaving the C-terminus from collagens Type I, II, and III, and it is identical to BMP-1 (which was misidentified as a bone morphogen).

Following cleavage and removal of the signal sequence, the proenzyme is separated into a propeptide sequence (left) and the active enzyme (right).

The activated enzyme acts as described above to establish stable cross-links and, modifies extracellular collagen substrates which promotes cellular migration, attachment, and cellular differentiation.    The propeptide segment controls cellular proliferation and prevents transformation by inhibiting signaling through a number of mechanisms downstream from Ras including Raf/MEK and PI3K/Akt, and the Wnt/β‑catenin pathways.

The LOX paralogs have a virtually identical structure for their catalytic domain, but their propeptide sequences are quite different^1,2.

As the catalytic sequences are virtually identical for all forms our inhibitor will likely be effective across all variants, while inhibiting the actions of the propeptide regions will vary.

LOX in Cellular Therapy

The difference in the actions of the propeptide and the activated enzyme provide an opportunity to engineer a cell delivery device that acts as an in situ bioreactor.  Technologies are known whereby a quantity of drug can be loaded into a hydrogel-based carrier that will act as a "drug depot" releasing a predetermined quantity of drug per unit time.  Since the propeptide is responsible for regulating the replication of the cells and the enzyme is responsible for regulating differentiation inhibition of the enzyme, but not the propeptide, will temporarily halt differentiation while cellular proliferation progresses. 

A proof of concept has already been demonstrated using a different inhibitor of LOX, albeit not one suitable for human use due to the toxicity of the agent.  Creating a biodegradable delivery device that inhibits LOX for a short period, say ten days, will allow undifferentiated cells expansion in the body precisely at the site of injury comparable to the best known ex vivo cell culture technologies, for a fraction of the cost, without the need for expensive capital investment, and without waiting two weeks to treat the patient.

LOX in Metabolic Disease

Our LOX inhibitor was designed as an oral drug to be administered to patients with fibrotic diseases.  Since each of our drug candidates attack collagen metabolism directly, our drug should be generally applicable to any disease where excess scar formation is a problem.  That includes end-stage renal disease, idiopathic pulmonary fibrosis, cardiomyopathy, scleroderma, and the one that will be discussed here, liver disease.

There is a rapidly growing incidence of NAFLD (non-alcoholic fatty liver disease) and its associated condition NASH (non-alcoholic steatohepatitis).  Fatty liver disease is the build up of small fat particles in the liver, largely as a result of poor dietary habits, lack of exercise and other lifestyle issues.

The precise incidence of fatty liver disease is difficult to establish because there are no obvious symptoms until the condition progresses.  A definitive diagnosis can only be made with a histological analysis of their liver tissue following a biopsy, or magnetic resonance imaging of their abdomen.  Neither are routine diagnostic procedures performed in the absence of other symptoms.

As the chart at left shows, there is clearly a genetic component as well since nearly 1 in 6 adults of Hispanic ethnicity have fatty liver disease compared with 1 in 3 African Americans.

NAFLD is often a progressive disease, with about half of the patients progressing to NASH over a seven year period.  NASH is quite similar except that enough liver tissue has become scarred at that point that a physician can feel the difference in elasticity when making a manual examination of the abdomen, and there may be changes in liver enzyme levels.

If NASH is not treated, an estimated 1 in 4 patients will progress to more serious fibrosis, cirrhosis, or hepatocellular carcinoma.

In animal experiments it was shown that Type I collagen break down products were elevated in those receiving LOX inhibitor treatment.  Presence of Type I collagen fragments is indicative of scar resolution, especially since there should be very little Type I collagen present in a normal liver. 

Two of the drug candidates underwent testing in a widely used assay in the pharmaceutical industry. The left side graph shows that at the 100μM concentration of the drug, deposition of collagen was reduced 96% versus control, and the fibrotic area was 63% smaller versus control.  This shows that the candidate drugs are powerful, and specific, inhibitors of the enzyme.

LOX in Cancer

LOX is known to play a role in various cancers, especially forms with ductal tumors such as breast and prostate cancer, and the LOXL-2 variant is known to promote metastasis.  These effects may be due to the four SRCR domains in the propeptide region which modulate the innate immune response.