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. 2022 Sep 14;12(1):15440.
doi: 10.1038/s41598-022-19634-2.

Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders

Sven Ruf #  1 , Sridharan Rajagopal #  2 , Sanjay Venkatachalapathi Kadnur  3 , Mahanandeesha S Hallur  3 , Shilpa Rani  3 , Rajendra Kristam  3 , Srinivasan Swaminathan  3 , Bharat Ravindra Zope  3 , Pavan Kumar Gondrala  3 , Indu Swamy  3 , V P Rama Kishore Putta  3 , Saravanan Kandan  3 , Gernot Zech  1 , Herman Schreuder  1 , Christine Rudolph  1 , Ralf Elvert  1   4 , Joerg Czech  1 , Swarnakumari Birudukota  3 , M Amir Siddiqui  3 , Niranjan Naranapura Anand  3 , Vishal Subhash Mane  3 , Sreekanth Dittakavi  3 , Juluri Suresh  3 , Ramachandraiah Gosu  3 , Mullangi Ramesh  3 , Takeshi Yura  3 , Saravanakumar Dhakshinamoorthy  5 , Aimo Kannt  6   7   8
Affiliations

Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders

Sven Ruf et al. Sci Rep. .

Abstract

Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Identification of tricyclic lead (1) from screening hits (A) and (B). IC50 on human NNMT are 1.6 µM (A), 0.18 µM (B) and 0.13 µM (1).
Figure 2
Figure 2
Other modifications in our lead series.
Figure 3
Figure 3
In-vitro activity of JBSNF-000028: Inhibition of recombinant (A) human, (B) mouse or (C) monkey NNMT as determined by fluorescence of an MNA derivative. (D) Inhibition of human NNMT as determined by LC/MS detection of MNA. (E) Inhibition of NNMT in U2OS cells as measured by LC/MS detection of MNA.
Figure 4
Figure 4
JBSNF-000028: Pharmacokinetics profile and target engagement. (A) Plasma concentration versus time profile of JBSNF-000028 in C57BL/6 mice. (B) Plasma levels of MNA confirming target engagement by JBSNF-000028 in C57BL/6 mice.
Figure 5
Figure 5
Efficacy of JBSNF-000028 in chronic DIO model. (A) Body weight changes (%) and cumulative energy intake in lean control animals and HFD fed animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (B) Fed blood glucose and fed plasma insulin profile of lean control animals and HFD fed animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (C) Plasma triglycerides, plasma LDL cholesterol, liver triglycerides, liver total cholesterol, MNA measurements in liver and visceral WAT samples of lean control animals and HFD fed animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (D) Oral glucose tolerance test (OGTT), (E) Area under the curve (AUC) for the OGTT. (F) Plasma insulin levels at t = 15 min. (G) Plasma insulin and (H) plasma glucose concentrations at t = 0 min. (I) HOMA-IR index. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.0001 and ****p < 0.0001 when compared with lean Control and $p < 0.05, $$p < 0.01, $$$p < 0.001, $$$$p < 0.0001 when compared with HFD Control. Two way ANOVA followed by Bonferroni’s post-hoc test (A, B, D); One way ANOVA followed by Bonferroni’s post-hoc test (C, E–I).
Figure 6
Figure 6
Efficacy of JBSNF-000028 in genetic model of type-2 diabetes (db/db mice). (A) Body weight changes (%) and cumulative energy intake in db/– control animals and db/db animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (B) Fed blood glucose and fed plasma insulin profile of db/– control animals and db/db animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (C) Plasma total cholesterol, plasma LDL cholesterol, plasma HDL cholesterol, MNA measurements in liver and visceral WAT samples of db/– control animals and db/db animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (D) OGTT profile, (E) Area under the curve (AUC), (F) plasma glucose and (G) plasma insulin levels at the time of the glucose bolus. (H) Plasma insulin levels at t = 15 min after the glucose bolus in the OGTT. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 when compared with db/– control and $$$$p < 0.0001 when compared with db/db vehicle control. Two way ANOVA followed by Bonferroni’s post-hoc test (A, B, D); One way ANOVA followed by Bonferroni’s post-hoc test (C, E–H).
Figure 7
Figure 7
Efficacy of JBSNF-000028 in ob/ob model. (A) Body weight changes (%) and cumulative energy intake in ob/– control animals and ob/ob animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (B) Fed blood glucose and fed plasma insulin profile of ob/– control animals and ob/ob animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (C) Plasma triglycerides, plasma total cholesterol, plasma LDL cholesterol, MNA measurements in visceral WAT and s/c fat samples of ob/– control animals and ob/ob animals treated with vehicle or JBSNF-000028 at 50 mg kg−1. (D) OGTT profile, (E) Area under the curve (AUC), (F) plasma glucose levels at the time of the glucose bolus. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 when compared with ob/– control and $$$$p < 0.0001 when compared with ob/ob vehicle control. Two way ANOVA followed by Bonferroni’s post-hoc test (A, B, D); One way ANOVA followed by Bonferroni’s post-hoc test (C, E, F).
Figure 8
Figure 8
Effect of 4-w treatment with JBSNF-000028 (50 mg kg − 1 bid) in NNMT knockout animals on HFD. (A) NNMT expression in white adipose tissue of wild-type and NNMT knockout mice. (B) Plasma MNA concentrations in wild-type or knockout animals with or without treatment with JBSNF-000028. (C) Plasma glucose concentrations under fed conditions before (filled columns) and after (hatched columns) four weeks of treatment with JBSNF-000028. (D) Oral glucose tolerance test after four weeks of treatment with JBSNF-000028. (E) Plasma insulin levels 15 min after oral glucose bolus in the glucose tolerance test. *p < 0.05, **p < 0.01; ***p < 0.001 versus the other indicated column. (D) *p < 0.05 wild-type vs knockout (vehicle-treated), ***p < 0.001 for JBSNF-000028 treated wild-type (dark blue) or knockout (light blue) animals vs. vehicle-treated wild-type mice. ###p < 0.001 for JBSNF-000028 treated wild-type (dark blue) or knockout (light blue) animals versus vehicle-treated knockout mice.
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